Biallelic expression of the IGF2 gene in human breast disease
Biallelic expression of the IGF2 gene in human breast diseaseAmanda H. McCann1,*,+, Nicola Miller1,+, Anne O'Meara2, Inge Pedersen1, Karina Keogh1, Thomas Gorey3 and Peter A. Dervan1
1Department of Pathology and the Biotechnology Centre, Laboratory I, G08, University College Dublin (UCD), Belfield, Dublin 4, Ireland, 2Our Lady's Hospital for Sick Children, Crumlin, Dublin 12, Ireland and 3Department of Surgery, UCD, Belfield, Dublin 4, Ireland
Received March 18, 1996;Revised and Accepted May 20, 1996
We examined the imprinting status of the insulin-like growth factor II gene (IGF2) in a series of 20 human breast disease samples to determine if disrupted imprinting (as evidenced by biallelic expression), was a demonstrable mechanism of altered gene expression. These samples included benign (n = 7) and malignant breast lesions (n = 13). Biallelic expression of IGF2 was detectable in 67% of benign and 60% of malignant informative breast lesions. Three informative reduction mastectomies displayed normal IGF2 imprinting. The presence of this alteration in human breast tissue is a novel finding, and may contribute to tumorigenesis, possibly by favouring an enhanced proliferative milieu, during which additional mutations could occur.
Genetic alterations in human breast cancer are well documented and include gene amplification of cellular proto-oncogenes and chromosomal deletions and/or point mutations in tumour suppressor genes (reviewed in 1 ). Another group of genes whose alteration could possibly underly the initiation or progression of tumorigenesis is an expanding family of genes known as imprinted genes, of which the insulin-like growth-factor II gene (IGF2) is a member (2 ,3 ).
The vast majority of autosomal genes are biparentally inherited, resulting in one paternal copy and one maternal copy, both of which are transcriptionally processed. With imprinted genes, however, even though there is biparental inheritance resulting in two copies of the gene at the DNA level, only one is expressed. The second copy is `silent' or imprinted (4 ). The expressed copy is governed molecularly by a gamete-specific tag resulting in exclusive or predominant expression either from the maternal or paternal copy (monoallelic expression). In the case of IGF2, this results in expression solely from the paternal allele in most tissues, whereas the maternal allele is `silent' (2 ,3 )[the exceptions in humans are liver (5 ), foetal choroid plexus and lepatomeninges (6 ) where IGF2 shows biallelic expression]. Considering that this `one copy only' dosage of imprinted genes is normal, altered gene dosage or disruption of the molecular systems involved in the correct identification and maintenance of the imprint could have deleterious effects on cellular function.
Two mechanisms involving alteration of the imprinting processing system have been suggested to play a role during tumorigenesis (7 ). One model proposes that inappropriate methylation (hypermethylation) and hence silencing of one copy of a tumour suppressor gene may occur (7 ). This could result from mutations in, or inappropriate activation of, imprintor genes (8 ,9 ). Aberrant silencing of one copy of a tumour suppressor gene requires only one additional event [probably loss of heterozygosity (LOH)] to render both copies non-functional, thereby fulfilling the criteria of Knudson (10 ). Hypermethylation as an alternative pathway for tumour suppressor gene inactivation has been demonstrated for the p16 (9 ), the von Hippel Lindau (VHL) syndrome (11 ) and the retinoblastoma (Rb1) (12 ) suppressor genes, lending credence to this hypothesis.
The other mechanism of altered imprinting, which in this case is applicable to IGF2, is a gene activation hypothesis(7 ). This involves reactivation of the silent maternal IGF2 allele (loss of imprinting, LOI), resulting in IGF2 expression from two active copies (biallelic expression) similar to paternal IGF2 heterodisomy. Several reports provide evidence that biallelic expression of IGF2 may play a role in human tumorigenesis. LOI of the IGF2 gene has been reported in a subgroup of Beckwith-Wiedemann syndrome (BWS) patients (13 ), a somatic overgrowth syndrome associated with a predisposition to a number of mesodermally derived embryonal tumours. LOI has also been reported at various frequencies in embryonal tumours from non-BWS patients such as Wilms' (14 ,15 ), rhabdomyosarcomas (16 ,17 ) and hepatoblastomas (18 ,19 ). Testicular germ cell tumours (20 ), choriocarcinomas(21 ), primary lung cancers(22 ) and cervical carcinomas (23 )also have demonstrable biallelic expression in a proportion of cases. IGF2 is a potent mitogen with possible autocrine and paracrine effects. Biallelic expression, resulting hypothetically in double the growth factor dosage, may explain not only the somatic overgrowth characteristics found in BWS patients, but may also play a role in tumour development (24 ).
In breast cancer, IGF2 is mitogenic for selected breast cancer epithelial cells in vitro (25 ). This, coupled with reports that IGF2 mRNA transcripts are present in both benign and malignant breast lesions at varying levels of expression (25 ,26 ) indicates that the IGF2 peptide may be involved in the growth regulation of breast tissue. Due to the fact that no gene rearrangement, gene deletion or gene amplification appeared to underly the differential levels of IGF2 mRNA transcripts in these studies (25 ,26 ), the aim of the present study was to investigate if biallelic expression of the IGF2 gene could be detected in a series of histologically benign and malignant breast lesions using an ApaI transcribed polymorphism (27 ) and PCR-based methodology.
We report that, of the informative breast lesions identified, 3/5 (60%) infiltrating ductal carcinomas and 2/3 (67%) benign lesions demonstrated biallelic expression of IGF2, while three informative reduction mastectomies had normal IGF2 imprinting in place (monoallelic). To our knowledge, this is the first report of biallelic expression of an imprinted gene, namely IGF2, in tissues from patients presenting with breast carcinoma and benign breast disease, and is only the third report of such an alteration in a common adult cancer (22 ,23 ). Based on previous studies in transgenic mice (28 ,29 ), this double dosage of IGF2 may play a role in prolonging cellular survival during which time other genetic mutations could accumulate (28 ). Alternatively, it may provide a `second signal', triggering the progression of cells already harbouring an oncogenic insult (28 ).
In total, 20 randomly selected breast lesions were investigated for biallelic expression of the IGF2 gene. Following DNA-PCR amplification of a 292 bp fragment encompassing an ApaI polymorphic site in exon 9 of the IGF2 gene, ApaI-HinfI restriction digestion identified informative samples. This was evident from the presence of two polymorphic bands of 256 and 231 bp in size (allele a and b respectively) (Fig. 1 ), indicating retention of both copies of IGF2. Using this previously described approach (30 ), heterozygosity was demonstrated in 40% (8/20) of the breast lesion samples. Examples of these cases and an informative metastatic adenocarcinoma of unknown primary origin are shown in Figure 1 . We also identified three informative reduction mastectomies using this approach. In a series of childhood cancers screened, 16% (4/25) of Wilms' tumours and 33% (4/12) of rhabdomyosarcomas were informative (data not shown). Two non-informative Wilms' tumours (WtnFand Wtn21) are also represented in Figure 1 . Total RNA was extracted from each of the heterozygotes and studied further.
To eliminate possible contaminating DNA in the RNA preparations, the samples were treated with DNase prior to reverse transcription and subsequent RT-PCR amplification. Similarly to previous studies (15 ,30 ), the use of intron-spanning primers (B and C) generated a 1.12 kb RT-PCR product, confirming the cDNA source as RNA. To determine allelic usage of the IGF2 gene, the ApaI transcribed polymorphic site was regenerated from the 1.12 kb RT-PCR product using primers A and R (27 )(primer A is denoted F in ref. 27 ), to yield a shorter nested PCR (nPCR) product of 236 bp in size which, on ApaI digestion and electrophoresis in 3% nusieve, allowed biallelic versus monoallelic expression to be assessed (Fig. 2 ). A monoallelic profile was identified as the 236 bp fragment (allele a) or a 173/63 doublet (allele b1/b2). A biallelic profile yielded all three fragments (Fig. 2 ). Whereas biallelic expression was clearly evident in some samples (MBI, Fig. 2 ), others displayed various levels of relaxation (BBB, BBE, MBF, Fig. 2 ). This has been described by others (22 ), and may be due either to low level expression of a previously imprinted allele (5 ) or to complete relaxation of IGF2 imprinting in a subpopulation of tumour cells (22 ).
Benign. Of the informative benign breast lesions (n = 3), two (BBB and BBE) showed biallelic expression for IGF2 (Fig. 2 , Table 1 ). Histologically, both showed epithelial hyperplasia. One was fibrocystic disease with ductal hyperplasia (epitheliosis), the other showed atypical lobular hyperplasia. The remaining benign lesion was a fibroadenoma (BB1D) retaining monoallelic expression from the 236 bp allele (allele a) in both the fibroadenomatous lesion itself (BB1D) and in adjacent pathologically normal tissue (BB2D) taken at a distance of 5 cm (Fig. 2 ). Finally, three informative reduction mastectomies were all monoallelic.
Ten informative childhood cancers were assessed for monoallelic or biallelic usage of IGF2. Of the four informative Wilms' tumours, 50% demonstrated biallelic expression. Of the four rhabdomyosarcoma heterozygotes, 50% had biallelic expression for IGF2 (data not shown).
In the present study, we examined allelic expression of the IGF2 gene in a series of adult breast lesions, using an ApaI transcribed polymorphism in exon 9 of the IGF2 gene (27 ). Of the informative cases, the normal imprinting profile (monoallelic status) was altered (biallelic) in a significant proportion of Wilms' (2/4; 50%) and rhabdomyosarcomas (2/4; 50%), similar to previous studies (14 -17 ). Importantly, of the breast cases examined, 2/3 (67%) benign and 3/5 (60%) malignant breast lesions demonstrated biallelic profiles. In contrast, normal imprinting was in place in three informative reduction mastectomies. To our knowledge, this is the first report to show that altered expression of an imprinted gene, namely IGF2, is present in double the allelic dosage in adult breast disease.
Two mechanisms may be responsible for this biallelic expression, either loss of imprinting (LOI), resulting in an active maternal copy in addition to an active paternal allele, or paternal IGF2 heterodisomy, resulting in two active paternal IGF2 copies. These two possibilities can only be distinguished by determining the parental origin of the alleles from blood samples, which as yet has not been feasible. Either way, however, both mechanisms hypothetically could result in twice the normal gene dosage of IGF2. Of those studies that have assigned parental origin to the IGF2 alleles, all cases with biallelic expression originated from an LOI mechanism (14 ,15 ).
Our finding of IGF2 biallelic expression in breast tissue is interesting. Firstly, in previous studies, structural genomic alterations of IGF2, such as gene rearrangement or amplification, could not explain the differential presence of IGF2 mRNA transcripts in the T47D, HBL100 epithelial cell lines, compared with non-expressing cultures (25 ). Neither could such alterations be detected in those breast carcinomas with as much as 20-fold differences of IGF2 mRNA levels (25 ,26 ). In the light of the present study, biallelic expression of IGF2 could, hypothetically, be responsible for these differential levels of IGF2. This finding, in conjunction with observations identifying the WT1 gene not only as a polymorphically imprinted gene in humans (31 ) but also as a transcriptional repressor of IGF2 (32 ), suggests (though speculatively) that lower levels of the WT1 product due either to monoallelic expression or due to LOH of one copy of the WT1 gene could possibly contribute further to the increased levels of IGF2.
Secondly, Northern analyses have detected IGF2 mRNA transcripts, not only in malignant and benign clinical breast tissues (25 ), but also in homogeneous epithelial (25 ) and fibroblast (stromal) cultures (33 ). This suggests that biallelic expression theoretically could originate from both cell types.
As to the biological role of IGF2 in breast development, and its possible role in tumorigenesis, evidence shows that IGF2 is mitogenic for breast tumour epithelial cells (25 ) and may function both in an autocrine and paracrine fashion in a proposed model of stromal/epithelial interactions (33 ). Direct evidence that biallelic expression may contribute functionally in the early stages of tumorigenesis comes from studies examining IGF2 allelic expression in an oncogene-driven multistage mouse model (RIPI-Tag2) of islet cell carcinogenesis (28 ,29 ). In the early stages of this model, the `proliferative switch' resulting in hyperproliferation and subsequent tumour development required co-activation of both IGF2 alleles, with each additively contributing to hyperproliferation, tumour growth and resultant tumour volume. In contrast, examination of tumour cell growth in RIPI-Tag2 mice with disrupted IGF2 function showed a reduction in tumour cell growth, reduced malignancy and a significant increase in the number of apoptotic bodies (28 ). In this last regard, IGF2 may play a role by prolonging cellular survival by inhibiting apoptosis(28 ). Certainly in cultured cells, its absence results in programmed cell death (34 ). The fact that we found biallelic expression of IGF2 in two fibrocystic cases with proliferative manifestations warrants further examination.
In addition to prolonging survival, altered IGF2 expression may act as a `second signal' in oncogene-induced tumorigenesis (28 ). In the RIPI-Tag2 studies (28 ,29 ), the Tag oncoprotein was expressed in all [beta] cells from embryonic day 9; however, the `proliferative switch' as a prerequisite to tumour development only occurred 3-4 weeks later. The coincidental activation of IGF2 at this later stage suggested that IGF2 may be providing a `second signal' favouring progression to a malignant phenotype. Whether increased expression of IGF2 is due to biallelic expression or other transcriptional events, elevated levels of IGF2 have been reported in several tumours, suggesting that it could, hypothetically, have served as a progression signal.
It is interesting that patients presenting with benign proliferative breast disease with epithelial proliferation have a slightly increased risk of subsequent malignancy, whereas those women presenting with fibroadenomas do not (35 ). In our informative benign cases, the two benign proliferative cases were biallelic, whereas the fibroadenoma was monoallelic. An investigation of a larger series of lesions will clarify the significance of IGF2 biallelic expression in these cases.
The 11p15.5 locus is a chromosomal region frequently affected by LOH in breast cancer (36 ). This, coupled with the fact that this region in humans harbours not only the imprinted IGF2 gene, but also the reciprocally imprinted H19 gene (37 ) with tumour suppressor gene activity (38 ), highlights the complexity of gene expression in this region. In addition, IGF2 may be controlled at the transcriptional level by the WT1 tumour suppressor gene product (32 ), and at the post-transcriptional level by the receptor for IGF2 (IGF2R) (39 ). The fact that both these genes may be subject to polymorphic imprinting in humans (31 ,40 ) indicates that such a possibility cannot be ruled out for IGF2. Of those studies that have reported on normal non-foetal tissue (5 ,14 ,17 ,18 ,22 ), IGF2 was monoallelic in all cases. In our reduction mastectomies, normal imprinting of IGF2 also appeared to be in place. Currently, we are examining an extended series of reduction mammoplasties to clarify whether IGF2 imprinting is genetically polymorphic.
In summary, a new finding in the present study is the identification of biallelic expression of the imprinted IGF2 gene in a certain proportion of breast lesions. This is a novel mechanism for altered gene expression in breast tissue and may lead to differential levels of mRNA. It could also explain why no underlying structural genomic alterations could be demonstrated in previous studies (25 ,26 ). It remains to be determined if this alteration originates from epithelial or stromal elements or both, but it does represent a mechanism that could, theoretically, lead to double the gene dosage of IGF2 with subsequent increased expression and protein levels. Due to the proposed autocrine and paracrine growth effects of IGF2 in breast tissue, this double dosage could play a vital role in enhancing the proliferative milieu of both the epithelial and fibroblast cell types. The finding of biallelic expression of the two benign proliferative cases in our study, and the presence of IGF2 mRNA transcripts in phenotypically normal fibroblasts derived from malignant epithelial cultures (33 ), suggests that this alteration could possibly play a role in the early stages of tumorigenesis. This is substantiated by the findings in transgenic mice that overexpression of IGF2 can cause mammary cancer (41 ).
Fresh tissue samples were snap frozen and stored at -70oC. Prior to nucleic acid extraction, separate samples from each specimen were set aside for the purposes of DNA and RNA extraction. There was no history of Beckwith-Wiedemann syndrome (BWS) among the Wilms' or rhabdomyosarcoma patients.
High molecular weight DNA was extracted by conventional methods. DNA-PCR reactions were performed on 1 [mu]g of genomic DNA essentially as described elsewhere (15 ,30 )using primers A and B (denoted P2 and P3 in ref. 15 ) which flank a transcribed ApaI polymorphism (27 ) in exon 9 of the IGF2 gene. The cycling conditions were as previously cited (30 ). The resultant 292 bp DNA products were digested with ApaI-HinfI, electrophoresed through 3% nusieve gels and stained with ethidium bromide. Tissue samples yielding polymorphic bands of 256 and 231 bp in size (allele a and b respectively), indicated heterozygosity for the ApaI polymorphism. In total, eight breast lesions, three reduction mastectomies and eight juvenile cancers (Wilms' and rhabdomyosarcomas) were informative using this approach and were studied further.
Total RNA was extracted from heterozygotes using the Ultraspectm II RNA isolation system (Biotex) and DNase treated. Following acid phenol-chloroform extraction and ethanol precipitation, 5 [mu]g of RNA was reverse transcribed with primer B to cDNA (Reverse Transcription System Promega). A reverse transcription PCR (RT-PCR) was performed on 1 [mu]l of first strand cDNA with primers B and C and the same PCR cycling conditions as for DNA-PCR to yield a 1.12 kb IGF2 RT-PCR product(30 ) which was confirmed by sequencing (data not shown). As these primers span an intron, DNA-specific PCR product (1.4 kb) could be detected separately from cDNA-specific product (1.12 kb) by agarose gel electrophoresis, and so made it possible to ensure that the nucleic acid source for the cDNA was RNA and not contaminating DNA. None of our RT-PCR preparations yielded a 1.4 kb genomic DNA band, indicating that there was no DNA contamination and that DNase treatment was successful. Due to the weakness of the 1.12 kb band in some samples, we used nested PCR (nPCR) on the 1.12 kb PCR products using primers A and R (27 ) (primer A is denoted F in ref. 27 ) to generate a shorter RT-PCR fragment of 236 bp harbouring the polymorphic site as previously published (27 ). Restriction digest analysis using ApaI revealed either monoallelic or biallelic usage of parental alleles. This was repeated for each sample. The ApaI alleles were designated allele a (236 bp) and allele b1/b2 (176/63 bp).
The present study was supported in part by the Health Research Board (HRB) Ireland. Special thanks to Florence Grehan of the Photography Department, Mater Hospital, the Audio Visual Centre (AVC), UCD, Dublin, and Dr Pat O'Mahony for help with the sequencing. We also thank Dr Deirdre Devaney for supplying some childhood tumours. Finally, thanks to Professor William Gullick and Dr David Croke for helpful discussions concerning this submission, and Geraldine O'Driscoll for invaluable secretarial assistance.
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