Differential gene expression profile reveals deregulation of pregnancy specific β1 glycoprotein 9 early during colorectal carcinogenesis
© Salahshor et al; licensee BioMed Central Ltd. 2005
Received: 21 February 2005
Accepted: 27 June 2005
Published: 27 June 2005
APC (Adenomatous polyposis coli) plays an important role in the pathogenesis of both familial and sporadic colorectal cancer. Patients carrying germline APC mutations develop multiple colonic adenomas at younger age and higher frequency than non-carrier cases which indicates that silencing of one APC allele may be sufficient to initiate the transformation process.
To elucidate the biological dysregulation underlying adenoma formation we examined global gene expression profiles of adenomas and corresponding normal mucosa from an FAP patient. Differential expression of the most significant gene identified in this study was further validated by mRNA in situ hybridization, reverse transcriptase PCR and Northern blotting in different sets of adenomas, tumours and cancer cell lines.
Eighty four genes were differentially expressed between all adenomas and corresponding normal mucosa, while only seven genes showed differential expression within the adenomas. The first group included pregnancy specific β-1 glycoprotein 9 (PSG9) (p < 0.006). PSG9 is a member of the carcinoembryonic antigen (CEA)/PSG family and is produced at high levels during pregnancy, mainly by syncytiotrophoblasts. Further analysis of sporadic and familial colorectal cancer confirmed that PSG9 is ectopically upregulated in vivo by cancer cells. In total, deregulation of PSG9 mRNA was detected in 78% (14/18) of FAP adenomas and 75% (45/60) of sporadic colorectal cancer cases tested.
Detection of PSG9 expression in adenomas, and at higher levels in FAP cases, indicates that germline APC mutations and defects in Wnt signalling modulate PSG9 expression. Since PSG9 is not found in the non-pregnant adult except in association with cancer, and it appears to be an early molecular event associated with colorectal cancer monitoring of its expression may be useful as a biomarker for the early detection of this disease.
FAP is characterized by the development of hundreds to thousands of adenomas throughout the entire colon and rectum which, if left untreated, progress to colorectal cancer [1, 2]. FAP, an inherited tumour predisposition, is caused by mutant alleles of the adenomatous polyposis coli (APC) gene and provides an opportunity to define critical early genetic events in the development of tumours . Early development of a large number of colon adenomas in this disorder indicates that mutations in the APC gene can be rate-limiting in adenoma development. The majority of colorectal tumours are sporadic in origin, however, they exhibit close similarities to tumours resulting in inherited colorectal cancer syndromes. Most sporadic colon adenomas and carcinomas also harbour APC gene mutations . The APC gene, which has been recognized as a gatekeeper of colorectal carcinogenesis, is one of the key components of the Wnt signalling pathway. Wnt signalling induces nuclear translocation of transcriptionally active β-catenin through interference with the β-catenin-destruction complex, composed of glycogen synthase kinase-3 (GSK-3α and β), Axin (Axin1 and 2) and APC. In the absence of a Wnt signal this complex efficiently earmarks cytoplasmic β-catenin for degradation through the ubiquitin/proteasome pathway [5, 6].
To identify the possible differences between different adenomas that either predispose to cancer or result in benign growths, we compared variations in gene expression between different adenomas and normal mucosa from the same patient with a germline mutation in the APC gene. The approach was designed to identify very early changes that occur during adenoma formation and to detect aberrant regulation of genes required for adenoma-carcinoma progression. Microarray-based expression profiling revealed that gene expression patterns between different adenomas are very similar but are different from normal mucosa. We describe the increased expression of a specific member of the pregnancy specific glycoprotein family and show that induction of this gene is a very early event that does not appear to be dependent on activation of β-catenin.
Adenomatous polyps, tumours and matched adjacent normal mucosal tissue samples from 18 FAP cases (germline APC mutations detected by standard techniques), 60 sporadic colorectal cancer cases, five liver metastases and one normal placenta, were obtained from University Health Network (UHN) human tissue bank and the Familial GI Cancer Registry at Mount Sinai hospital, in compliance with each Institutional Review Board. Colorectal cancer cell lines; SW620, SW480, LoVo, RKO, SW1417, LS1034 and MCF12A were purchased from ATCC and grown in media recommended by the distributor. Total RNA samples from normal ovarian, prostate, colon, breast and placental tissues were purchased from Ambion and Clontech. RNA was extracted from cell lines and tissue samples using an RNAeasy kit (Qiagen). Tissues were processed for RNA extraction, in situ hybridization or immunohistochemistry analysis.
Microarray procedure and data analysis
cDNA microarrays consisting of 19,200 human gene clones were employed to explore the variation in gene expression between adenoma and normal mucosa. Microarray slides were obtained from the University Health Network Microarray Centre (UHN, Toronto, Canada). Protocols used for array hybridisation were as published on the UHN Microarray Centre web page http://www.microarray.ca/support/proto.html with some modifications. Briefly, 5 μg total RNA extracted from normal mucosa or adenoma was labelled with Cy5 and, a reference total RNA pool was labelled with Cy3. The reference RNA used was composed of total RNA from 10 human cell lines (Stratagene) which hybridize to the maximum number of spots on the array. The signals obtained from reference RNA have been used for normalisation of experimental samples. Microarray hybridization was carried out in a hybridization chamber humidified with 2XSSC. Labeled cDNA was dissolved in 80 μl of the hybridization buffer, denatured at 95°C for three minutes in a thermal cycler, and applied on the microarray slides. Microarrays were incubated overnight at 37°C. Post-hybridization washing was performed by serial incubations in buffers with decreasing SSC and SDS concentrations, at 50°C. At least two replicates including dye switches were performed for each experiment to account for possible dye labelling and hybridization bias. Relative expression was assessed by a two-colour hybridization experiment. Slides were scanned using either an Axon GenePix 4000A (Axon) or ScanArray 4000 Scanner (Packard BioScience). The scanned 16-bit TIFF images were quantified using QuantArray software (Packard BioScience). The quantified data files were transferred to a GeneTraffic microarray database and analysis system (Iobion Informatics, Stratagene) with a complete annotation of experiments based on the current MIAME standards for microarray experiments http://www.mged.org. Each hybridization dataset was filtered and spots that did not pass the quality criteria in both channels were excluded from further analysis. The lowess subarray normalization which uses a local weighted smoother to generate an intensity-dependent normalisation function was applied to each hybridization. The normalised log2 ratios were used for statistical analysis. Data were analysed by both SAM software (Significance Analysis for Microarrays) and the statistical program integrated into GeneTraffic 2.8. Genes exhibiting a consistent 2-fold or more up- or down regulation with a p value of <0.05 were considered significant.
Semi-quantitative and quantitative RT-PCR
Analysis of PSG9 sequence variants in tumors
PSG9 isoforms expressed in normal placenta, SW620 and LoVo colon cancer cell lines and two sporadic colorectal cancer cases, were PCR amplified. Different isoforms were separated on a 2% agarose gel and purified by PerfectPrep gel cleanup kit (Eppendorf). The purified DNA products were cloned into pCR®II-TOPO vector. Clones were sequenced in both directions using T7 and T3 primers using an ABI sequencing system (Applied Biosystems). Sequence variation was examined using GeneJockey II software (Biosoft).
Northern blot analysis
PSG9 expression patterns were analysed using random primed radiolabeled full-length PSG9 cDNA. A 2 kb fragment was digested from a cDNA clone (GeneBank Accession No. 196828) by PAC I and EcoRI restriction enzymes, gel purified and then radiolabeled with [α-32P]dCTP using a T7 Quik Prime system (Pharmacia Biotech). Northern blot analysis was performed based on standard techniques .
RNA in situ hybridisation
RNA in situ hybridization (RISH) was used for the examination of PSG9 and PSG2 mRNA expression. Nonradioactive RISH was applied to frozen or paraffin-embedded sections using digoxigenin (DIG)-labelled copy RNA (cRNA) probes. Different cDNA fragments corresponding to human PSG9 (GenBank 196828) were amplified by PCR using sense and antisense primers containing either T7, T3 or SP6 promoter sequences, respectively. PSG2 riboprobes were amplified from a human placental RNA pool (Clontech). The locations of primers and oligoprobes sequences are indicated in figure 1. For cRNA probe synthesis, purified PCR products were used. The transcripts were labelled with DIG-labelled nucleotides, DIG RNA labelling kit and either T7, T3 or SP6 RNA polymerase (Roche Applied Science) to produce DIG-labelled riboprobes. Primers used for amplification and synthesis of each cRNA probe were: PSG9-E2; Sense 5'-T GCC GAA GTC ACG ATT GAA G-3', Anti-sense: 5'-GGA TGC GTT GGA ATA TAC TGT TTC T-3', PSG9-E4-5; Sense: 5'-A TGT CTT AGC CTT CAC CTG TG-3', Anti-sense: 5'-AGT GCC GGT GGG TTA GAT T-3' PSG2; Sense: 5'-GTC CAG ACC TCC CCA GAA T-3', Anti-sense 5'-AGG CTG CTA TGT TGG ATT AAG GAG AG-3'. PCR conditions are available upon request. Seven-μm-thick sections of paraffin-embedded or fresh frozen tissue were cut, fixed in 1XPBS (phosphate-buffered saline) containing 4% paraformaldehyde. A standard in situ hybridization technique was used with some modifications. Images were analysed by light microscope (Leica).
Western blot and immunohistochemical analysis
To detect β-catenin and β-actin, cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris pH 8.0, 1 mM EDTA). Twenty μg protein were loaded onto 8% SDS polyacrylamide gels, separated and transferred to a nitrocellulose filter by semidry transfer. Western blotting were performed using a standard protocol. The antibody dilutions were as follows; primary antibodies β-catenin (1:1000; Transduction Laboratories) and β-actin (1:2000; Abcam), secondary monoclonal antibody (1:5000; BioRad). Standard immunohistochemistry (IHC) was carried out on formalin-fixed, paraffin-embedded or fresh frozen sections.
Wnt signalling stimulation and reporter gene assay
RKO cells were treated over night with 50 nM Wnt3a recombinant (R&D systems) or 10 μM Kenpaullone (Calbiochem) an inhibitor of GSK3  prior to RNA or protein extraction. RKO cells transfection was performed in six-well plates at the density of 1.5 × 105 cells/well with Lipofectamine 2000 reagent (Invitrogen). Cells were transfected with either Super8XTOP- or 8XFOPFlash and β-galactosidase constructs  and were assayed for luciferase activity 23 hrs post-transfection/treatment. The luciferase activity was measured and quantified in a luminometer using a chemiluminescent reporter gene assay system for the combined detection of luciferase and β-galactosidase as recommended by manufacturer (Applied Biosystems). The β-galactosidase was used to normalize luciferase units in each transfection.
Gene expression profiling
Genes upregulated in all adenomas compared to normal mucosa (p < 0.05).
pregnancy specific beta-1-glycoprotein 9
protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform
integrin, alpha 1
neural precursor cell expressed, developmentally down-regulated 8
aminolevulinate, delta-, synthase 2 (sideroblastic/hypochromic anemia)
jagged 1 (Alagille syndrome)
ribonuclease, RNase A family, 4
X-box binding protein 1
cell division cycle 42 (GTP binding protein, 25 kDa)
hypothetical protein MGC8407
nucleoporin 98 kDa
cyclin-dependent kinase inhibitor 1A (p21, Cip1)
interleukin 1 receptor, type II
phosphatidylinositol-4-phosphate 5-kinase, type I, beta
TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor
RAB25, member RAS oncogene family
anaphase-promoting complex subunit 7
mitochondrial carrier homolog 2 (C. elegans)
hepatocellular carcinoma-associated antigen 112
adenovirus 5 E1A binding protein
hypothetical protein MGC29898
hypothetical protein BC001584
follicular lymphoma variant translocation 1
Rho GDP dissociation inhibitor (GDI) alpha
vaccinia related kinase 3
major histocompatibility complex, class II, DP alpha 1
WAP four-disulfide core domain 2
G protein-coupled receptor 161
SLAM family member 8
hypothetical protein BC011630
Genes downregulated in all adenomas compared to normal mucosa (p < 0.05).
glial fibrillary acidic protein
clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2
vitamin D (1,25- dihydroxyvitamin D3) receptor
matrix Gla protein
insulin-like growth factor binding protein 2, 36 kDa
protein kinase C, epsilon
cytochrome c oxidase subunit IV isoform 1
apolipoprotein B (including Ag(x) antigen)
baculoviral IAP repeat-containing 4
integrin, alpha E (antigen CD103, human mucosal lymphocyte antigen 1; alpha polypeptide)
growth arrest-specific 1
GATA binding protein 3
vascular endothelial growth factor B
adiponectin receptor 2
RUN and FYVE domain containing 2
low density lipoprotein receptor-related protein 8, apolipoprotein e receptor
serine hydroxymethyltransferase 2 (mitochondrial)
TXK tyrosine kinase
hypothetical protein LOC51315
UDP-glucose ceramide glucosyltransferase-like 2
hypothetical protein MGC5178
I-kappa-B-interacting Ras-like protein 2
solute carrier family 35, member F2
hypothetical protein FLJ33761
zinc finger protein 198
phosphoprotein associated with glycosphingolipid-enriched microdomains
hypothetical protein FLJ40432
hypothetical protein BC013035
Ras-associated protein Rap1
hypothetical protein FLJ20360
hypothetical protein from EUROIMAGE 1967720
leader-binding protein 32
SATB family member 2
CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 1
oxysterol binding protein-like 2
hypothetical protein FLJ38991
hypothetical protein MGC12972
zinc finger protein 496
hypothetical protein FLJ35936
chromosome 14 open reading frame 141
sphingosine-1-phosphate phosphotase 2
hypothetical protein DKFZp313N0621
hypothetical protein LOC285550
hypothetical protein MGC52498
Genes differentially expressed within different adenomas compared to corresponding normal mucosa.
growth arrest-specific 1
insulin-like growth factor binding protein 2
microtubule-associated protein, RP/EB family, member 3
zinc finger, FYVE domain containing 20
hypothetical protein FLJ11848
PSG family member expression
Seventeen PSG clones are represented on the 19K human chip used in these studies: four clones represent PSG1, one for PSG3, two PSG4, two PSG5 clones, two PSG6, three PSG11, and three clones are assigned as PSG9. To examine why only one of the PSG9 clones on the cDNA chip indicated differential expression between normal mucosa and polyps, we re-sequenced all the clones representing PSG9. The sequencing results showed that only clone 196828 contained a full-length PSG9 cDNA. This clone represents the largest transcript of PSG9. Because of the high homology (>90%) between different PSG genes and possible cross-hybridization and non-specific binding of primers to different PSGs and also to different transcripts of PSG9, all primers were designed based on the sequence of this clone (Fig. 1a).
Expression of PSG9 in normal and cancer cells
Next, we examined whether the PSG9 expressed in tumours was different from PSG9 expressed by placental cells during pregnancy. The coding sequences of all PSG9 variants of two colon cancer cell lines (SW620 and LoVo) and two sporadic colorectal cancer cases were cloned and sequenced. Two sequence variations were found in the non-coding region, while no changes were found in the coding region of PSG9. These results indicate that the PSG9 proteins expressed by placental cells and tumour cells have similar sequences and might have similar function, however, the level of expression of each PSG9 variant differed from those found in placenta.
Effect of Wnt signal activation on PSG9 expression in the absence of APC mutation
In this study, we have shown that PSG9 is ectopically expressed in colorectal cancer and this is most likely APC dependent, since abnormal expression can be detected as early as in normal appearing epithelial cells and adenomas of the FAP cases which carry APC germline mutations, while corresponding normal tissue in sporadic colorectal cancer tumors lack PSG9 expression. Given the increased expression of PSG9 in the mucosal cells of FAP patients that displayed lower β-catenin stabilisation compared to sporadic colorectal tumours in our study, it is possible that PSG9 is not directly regulated by the β-catenin/Wnt signalling pathway and that other molecules that regulate PSG9 expression are altered as a consequence of APC mutation. Notably, deregulation of PSG9 is detectable as early as in mucosa that appears histologically normal in FAP cases with APC germline mutations, suggesting that the dose and gradient of APC is important in PSG9 regulation. Functional analysis of APC protein has revealed a broad spectrum of activities for this molecule [22, 23]. In normal-appearing epithelial cells in FAP, PSG9 was expressed mostly on the apical surface, while in tumours, expression was detected from the top to the base of crypts. Early expression of PSG9 even before adenoma formation at the top of the crypts in FAP cases, suggests that transformation process starts in cells at the top of the crypts which then gradually move downward (Fig. 4d). These observations are consistent with the "top-down" morphogenesis model of colorectal cancer .
PSGs exhibit sequence similarity to the carcinoembryonic antigen (CEA) family which, in turn, is a member of the immunoglobulin (Ig) superfamily. The CEA gene family can be divided into three subgroups; the CEA subgroup (12 genes), PSG subgroup (11 genes) and a pseudogene subgroup (6 genes) . CEA is a widely used tumour marker, the main clinical utility of which is in monitoring clinical course of colorectal carcinoma after surgical resection . Contrary to its name, CEA is expressed in normal adult tissue, as well as during fetal development. The role of CEA in normal human physiology is not well understood. Based on its structure, a number of functions have been suggested, including intracellular cell adhesion, signal transduction or signal transduction regulation. PSGs are secreted proteins and, in contrast to CEA, most PSGs are not expressed in normal colon epithelial cells. The main site for PSG production is the placental syncytiotrophoblasts during pregnancy . Although PSGs were discovered more than three decades ago, their function is still unknown and the receptor(s) for these proteins has yet to be identified. Most PSGs have an RGD (Arg-Gly-Asp) motif in a conserved region in the N-terminal domain which suggest that these genes may function as adhesion recognition signals for integrins .
Immune privilege of cancer cells
Several studies suggest that T-cells are capable of recognizing and responding to tumours in experimental conditions, yet most tumours are able to escape detection by the immune system. A common characteristic of cancer and the placenta is their ability to avoid immune reactivity. The capability of cancer cells to sidestep the body's immune reaction, is believed to be partly aided by the protective coating of the cells, which is largely composed of glycoproteins . PSGs are heavily glycosylated proteins and the primary amino acid sequence is masked by the sugar modification which helps in avoid specific antibody recognition . It has been suggested that PSG production by trophoblasts regulates maternal immune response to the fetus. It is also possible that PSG9 expressed by pre-malignant and cancer cells protect tumours from recognition by the body's immune defences. Another similarity between the respective milieu where both placenta and adenomas develop is their low-oxygen environment. Trophoblast invasion and placental development during the first trimester occurs in a low-oxygen environment, as the blood flow to the intervillous space is not yet established . We found lower levels of beta hemoglobin (HBB) RNA in adenomas compared to normal mucosa in this study (Table 1) as well as in another gene profiling study we have performed (manuscript in preparation).
Role of PSG9 in cancer
Most of the PSG subgroup (11 genes) are expressed. However, the existence of allelic variants with a stop codon in the N-domain of some PSGs, indicates that some individuals may not express all members of the PSG family . The possible involvement of PSGs in cancer and their genetic variation may in part explain phenotypic divergence that exists in cancer cases with otherwise identical germline mutations. The RGD sequence motif in the N-terminal domain of most PSGs is also present in a variety of extracellular matrix proteins that bind to integrin receptors such as fibronectin and vitronectin . It has been hypothesized that the PSGs (like most Ig superfamily members) are involved in adhesion/recognition processes. Another possibility is that upregulation of PSG9 might favour tumour development by causing a reversion of the monolayered adult colonic epithelium to an embryonic multilayered arrangement.
Our results provide strong evidence that PSG9 deregulation in cells occurs early during adenoma-carcinoma formation. High levels and early deregulation of PSG9 in adenomas, as well as normal mucosa in some FAP cases, indicates the potent role of APC germline mutations that are often found in these cases. The precise role of PSG9 in carcinogenesis remains to be determined. However, early-onset over-expression of PSG9 in different types of cancer suggests that this gene may be considered as a valuable biochemical tumorigenesis marker. To elucidate whether the frequency of occurrence of elevated PSG9 could have clinical significance, further analysis of serum levels of PSG9 and also other PSGs are warranted. Since PSG9 is not found in the non-pregnant adult except in association with cancer, it may be useful as a biomarker for the early detection of cancers of various types.
familial adenomatous polyposis
adenomatous polyposis coli
pregnancy specific glycoprotein
statistical analysis of microarrays.
This work was supported by the National Cancer Institute of Canada funded by the Canadian Cancer Society and Canadian Institutes of Health Research. SS appreciates the support of the Helena Lam fellowship. We thank Drs. Vogelstein, Kinzler and Moon for cDNA constructs, Dr. Hung for stable cell line, and Dr. Redson for support.
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