Loss of Imprinting of IGF2: A Common Epigenetic Modifier of

Intestinal Tumor Risk

Atsushi Kaneda and Andrew P. Feinberg

Division of Molecular Medicine, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland

Abstract

Epigenetic alterations in cancer occur at least as commonlyas genetic mutations, but epigenetic alterations could occursecondarily to the tumor process itself. To establish a causalrole of epigenetic changes, investigators have turned togenetically engineered mouse models. Here, we review arecent study showing that a mouse model of loss ofimprinting (LOI) of the insulin-like growth factor II gene(Igf2), which shows aberrant activation of the normallysilent maternal allele, modifies the risk of intestinalneoplasia caused by mutations of the adenomatous poly-posis coli (Apc) gene. This increased risk corresponds to theapparent increased risk of colorectal cancer in patientswith LOI of IGF2 . The model suggests that preexistingepigenetic alterations in normal cells increase tumor riskby expanding the target cell population and/or modulatingthe effect of subsequent genetic alterations on these cells,providing a novel idea for cancer risk management. (CancerRes 2005; 65(24): 11236-40)

Introduction

Epigenetics is the study of cellular information other than theDNA sequence itself, which is heritable in cell progeny andinvolves modification of DNA or its associated proteins. Epige-netic changes include DNA methylation, a covalent modificationof cytosine, and post-translational modifications of histone tails,such as acetylation, methylation, and phosphorylation (1). Epi-genetic alterations have been increasingly recognized as animportant mechanism in tumorigenesis since their discovery inhuman tumors in 1983 (2, 3). Genomic imprinting is an epige-netic modification of a specific parental chromosome in thegamete or zygote, leading to parental origin-specific differentialexpression of the two alleles of a gene in somatic cells of theoffspring (3).The insulin-like growth factor-II gene (IGF2), an imprinted

gene with parental allele expressed and maternal allele silenced, isan important autocrine growth factor in tumors due to itsmitogenic and antiapoptotic functions mediated by the type Ireceptor (IGF1R; refs. 4, 5). Increased expression of IGF2 is foundfrequently in a wide variety of malignancies, including colorectal,liver, esophageal, and adrenocortical cancer, as well as sarcomas(6). Paracrine signaling by IGF2 also plays a role in tumorsincluding breast cancers, as abundant expression of IGF2 is found

in stromal fibroblasts surrounding malignant breast epithelialcells (7).Although the downstream effectors are similar in signaling by

insulin, IGF1, and IGF2, the receptors are different, with insulinacting through the insulin receptor, and IGF1 and IGF2 actingthrough IGF1R (5). These signaling pathways are reviewedelsewhere (8, 9) and are briefly summarized here. IGF1R tyrosinekinase, when activated by ligand binding, phosphorylates severalsubstrates, such as the insulin receptor substrates IRS-1 andIRS-2. Phosphorylated IRS recruits the phosphatidylinositol 3-kinase (PI3K), resulting in increase of phosphatidylinositol-3,4-diphosphate and phosphatidylinositol-3,4,5-triphosphate in thecytoplasmic membrane and in activation of phosphoinositide-dependent kinases and protein kinase B/AKT. AKT phosphor-ylates a wide range of target proteins, including glycogensynthase kinase 3h (GSK3h), mouse double minute 2 (MDM2),bcl-associated death promoter (BAD), and mammalian target ofrapamycin (mTOR). GSK3h degrades cyclin D1, and inactivationof GSK3h by phosphorylation leads to accumulation of cyclinD1. MDM2 binds and degrades p53, and phosphorylation ofMDM2 increases its translocation to the nucleus and p53degradation. BAD is a member of the BCL-2 family, andphosphorylation of BAD prevents its positive regulation of cellapoptosis. mTOR regulates protein translation through activa-tion of p70 S6 kinase (S6K), translational enhancer of mRNAswith 5V-polypirimidine tracts, and through inhibition of eIF4Ebinding protein-1 (4EBP1), translational repressor of mRNAswith 5V-CAP sequences, such as cyclin D1 and MYC. This PI3K/AKT pathway is implicated in gene expression, cell survival, andgrowth signals. Activation of receptor tyrosine kinase alsotriggers another pathway. Growth factor receptor-bound protein2 (GRB2) binds to the receptor through its Src homologydomain SH2 and forms a complex with guanine nucleotideexchange factor SOS through its SH3 domains, resulting inactivation of Ras, Raf, and mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase (ERK) kinase(MEK). MEK phosphorylates MAPK/ERK, leading to transloca-tion of MAPK/ERK to the nucleus and activation of varioustarget proteins, such as ETS domain transcription factor ELK1and MYC. This Raf/MEK/ERK pathway is implicated in cellproliferation (8, 9).Loss of imprinting (LOI) of IGF2 , or aberrant activation of the

normally silent maternally inherited allele, was first described inWilms tumor, a childhood kidney cancer (10, 11), and later inmany common adult malignancies, including colorectal cancer(3, 12). What was unusual about LOI in colorectal cancer wasthat when it was present in tumors, it was also present inadjacent normal mucosa, albeit more commonly in patientswith colorectal cancer (12). A systematic analysis of 172 patientspresenting to a colonoscopy clinic showed a 4.7-fold increasedlikelihood of LOI among patients with past or present colorectalneoplasia, and a 5.2-fold increased likelihood of LOI among

Requests for reprints: Andrew P. Feinberg, Departments of Medicine, Oncology,and Molecular Biology and Genetics, Johns Hopkins University School of Medicine,Ross 1064, 720 Rutland Avenue, Baltimore, MD 21205. Phone: 410-614-3489; Fax: 410-614-9819; E-mail: afeinberg@jhu.edu.

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-2959

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patients with a positive family history of colorectal cancer amongfirst-degree relatives. This increased apparent risk was with LOI inperipheral blood lymphocytes, which when present also occurred inthe colonic mucosa, suggesting that a potentially geneticallydetermined stringency in the maintenance of imprinting wasfamilial and associated with colorectal cancer risk (13). Theassociation of LOI with colorectal neoplasia has subsequently beenconfirmed by two groups. The first study showed a 5-fold increasedlikelihood of LOI in normal colonic mucosa with colon adenomaformation (14). The second was an independent replicate in amixed racial/ethnic population, which showed a 5-fold increasedlikelihood of LOI in normal peripheral blood lymphocytes with thepresence of colorectal polyps or cancers (15).One of the hallmarks of epigenetic changes in cancer is that

they lead to a change in the level of expression of the affectedgene rather than a DNA sequence change. Proving that anepigenetic change plays a causal role is difficult, because theepigenetic alterations could arise secondarily in the tumor. Tocircumvent this problem, investigators have developed mouse

models to test the epigenetic hypothesis. For example, a mousecarrying a hypomorphic mutation for DNA methyltransferase I(Dnmt1 ) shows that DNA methylation is a modifier oftumorigenesis. Hypomethylation reduces the frequency of intes-tinal tumors in Min mice with a mutation in the adenomatouspolyposis coli (Apc) gene (16), as well as in Mlh1-deficient mice(17). However, the Dnmt1 hypomorphic mice show increasedlymphomagenesis either alone or when crossed with Mlh1-deficient mice (17, 18).

Mouse Model of Loss of Imprinting of Igf2 inIntestinal Neoplasia

LOI in human tumors leads to only a 2- to 3-fold increase of IGF2levels in both normal and tumor tissues (14, 19). Previous models ofIgf2 in cancer involved a substantial overexpression of Igf2 , whichmight act independently in tumorigenesis (20, 21). Our goal was toreproduce as physiologically as possible the double dose of IGF2expression caused by LOI.

Figure 1. Mouse model of LOI of Igf2 in intestinal neoplasia. Top left, in LOI(�) mice (H19+/+), the maternal H19 allele is expressed (red arrow ) with its differentiallymethylated region (DMR) unmethylated (white circles), and the maternal Igf2 allele is silenced. Also in LOI(�) mice, the paternal H19 allele is silenced with itsdifferentially methylated region methylated (red circles ), and the paternal Igf2 allele is expressed (blue arrow ). In LOI(+) mice (H19�/+), the maternal H19 anddifferentially methylated region are replaced with neo , and Igf2 is activated. Top right, female mice heterozygous for the H19 deletion were crossed with male Min mice(heterozygous for an Apc mutation) to produce the four different genotypes shown. Tumors were analyzed in LOI(�) and LOI(+) Min mice, and normal tissues wereanalyzed in LOI(�) and LOI(+) mice with and without Apc mutation. Data from ref. 22. Bottom, a model of the effect of LOI on small intestine. In LOI(�) mice, theprogenitor compartment (yellow within the crypts ) and the differentiated compartment in the villi (brown ), with the Paneth cells (white ). In LOI(+) mice, the progenitorcompartment is larger and also altered epigenetically (orange ). Finally, adenomas (red ) arise from this altered epithelium but are capable of differentiation given theirprogenitor cell origin.

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Our mouse model was designed by crossing female H19 deletionmice with male Apc Min mice (ref. 22; Fig. 1A-B). When deletion ofH19 and its upstream differentially methylated region is inheritedfrom mother, the offspring shows activation of the normallysilenced allele of Ig f2 (i.e., biallelic expression of Ig f2 by LOI;ref. 23; Fig. 1A). In Min mice, intestinal tumors are developed whena mutation of the Apc gene is inherited on one allele, and Min isconsidered a good model of human colorectal cancer because itinvolves a genetic mutation of the gatekeeper gene in humancolorectal cancer (24).When we crossed the female H19 deletion mice with male Min

mice, we found the desired 2-fold increase of Ig f2 in the normalintestinal tissue and tumors of LOI(+) mice compared with LOI(�)mice (22). The numbers of adenomas, surface area of adenomas,and number of adenomas per unit area of intestine in LOI(+) Minmice were increased 2.2-fold (P < 0.0001), 2.5-fold (P < 0.001), and1.9-fold (P < 0.0001), respectively, compared with LOI(�) Min mice(22). These data provide strong experimental evidence to supportthe data from human studies (13, 14), linking LOI of IGF2 tocolorectal cancer risk.In addition to the increase in tumor incidence in this model,

LOI(+) mice showed a shift of maturation of intestinal epithe-lium to a more undifferentiated state (Fig. 1C). This was appa-rent both in the length of intestinal crypts, the location of thestem cell compartment in intestinal epithelium, as well as in thenumber of cells with positive staining for progenitor cell mar-kers, such as Musashi1 and Twist (22). We also found a similarshift in maturation of intestinal epithelium using mice with amutation in the H19 imprinting regulatory region rather thanH19 deletion (22). Consistent with an effect of LOI on normalepithelium, the ratio of microadenomas to macroadenomas wasnot changed comparing LOI(+) Min mice with LOI(�) Min mice;that is, the rate of tumor initiation rather than tumorprogression was increased (22).Expansion of a progenitor cell population induced by LOI has

also been shown in human tissues. Patients with LOI showed astatistically significant increase in staining with progenitor cellmarkers in their colon (22). In addition, patients with Beckwith-Wiedemann syndrome, an overgrowth syndrome predisposingto Wilms tumor of the kidney, show focally expanded nephro-genic precursor cells termed perilobar nephrogenic rests (PLNR;

ref. 25). Similar PLNR are also found in about half of thekidneys of patients with sporadically occurring Wilms tumors,specifically those with LOI of IGF2 , and the PLNR themselvesalso undergo LOI (19). The Wilms tumors with LOI show anapproximate doubling of IGF2 expression (19), similar to thatinduced in the intestine in our mouse model. Thus, an epige-netically induced doubling of IGF2 seems to have the capacityto alter the balance between progenitor and differentiatedcells in diverse tissues, increasing the risk of malignancy severalfold.In addition to epigenetic mechanisms, genetic changes [e.g.,

inhibition of Bmp (26) and inactivation of Muc2 (27) or p27(28)] may alter this balance (Table 1). Inhibition of Bmp , agenetic model of hereditary juvenile polyposis, causes abundantectopic crypt formation (26). Inactivation of Muc2 or p27kip1

causes intestinal tumors, associated with increased cell prolif-eration and decreased apoptosis in normal intestinal epithelium,and decreased goblet cell differentiation (27, 28). Thus,expansion of progenitor cell populations may be a commonmechanism increasing cancer risk, and there may be multipleways to get to this end point.A second potential mechanism by which LOI might affect

intestinal tumor risk is by modulating the effect of APC muta-tion. APC is a cytoplasmic protein that interacts with both GSK-3h and h-catenin, promoting degradation of h-catenin. Mutationof APC increases the h-catenin and subsequent relocation of h-catenin to the nucleus, where accumulated h-catenin forms acomplex with TCF4, causing increased transcriptional activationof target genes, such as MYC , leading to increased cell proli-feration (29).In vitro transformation caused by deregulated MYC expression

requires exogenous growth factors, such as IGF2, IGF1, andplatelet-derived growth factor. Up-regulation of MYC inducesapoptosis in the absence of serum, but addition of any one ofthese growth factors suppresses MYC-induced apoptosis in vitro(30). IGF2 also causes relocation of h-catenin to the nucleus in vitroand activates transcription of target genes of the h-catenin/TCF4complex (31). Thus, the effects of APC mutation, such as activationof h-catenin pathway and MYC-induced transformation, might bemodulated by increased expression of IGF2 . Thus, LOI of IGF2 mayincrease cancer risk both by expanding the progenitor cell

Table 1. Genetic and epigenetic mouse models of intestinal tumors involving altered differentiation in the normal epithelium

Mouse model Target gene (reference) Frequency in normal human tissue Dedifferentiation phenotype

Genetic mouse models

Noggin transgene Bmp inhibition (26) Rare Ectopic crypt formation

Muc2�/� Muc2 inactivation (27) Rare Elongation of crypt

Decreased goblet cell differentiation

Increased proliferationDecreased apoptosis

p27�/� p27 inactivation (28) Rare Decreased goblet cell differentiation

Increased proliferationDecreased apoptosis

Epigenetic mouse model

H19�/+ Ig f 2 LOI (22) Common Elongation of crypt

Increased staining of progenitor cell markers

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compartment and by augmenting h-catenin signaling of genes,such as MYC .

Conclusion

We have proposed that epigenetic alterations, in addition toserving as a surrogate of genetic change, can precede thedevelopment of cancer and increase the probability of cancerwhen genetic changes arise (32). LOI in colorectal cancer is anexample of this model, in that LOI of IGF2 in normal cells iscommonly associated with colorectal cancer in the humanpopulation (12–15) and plays a causal role in increasing the riskof intestinal neoplasia in a mouse model (22). Another potentialexample is p16 INK4 promoter methylation in some normal humanbreast epithelial cells, which when cultured acquire tumor-relatedproperties, such as chromosomal alterations and cyclooxygenase-2overexpression (33).This epigenetic model suggests a novel and important

approach to cancer prevention. One might attempt to down-modulate the increased signaling caused by LOI of IGF2 inpatients before their developing neoplasms. It has been shownthat modification of IGF1R signaling reduces cancer cell growth[e.g., by addition of neutralizing antibodies (34), an excessamount of binding proteins (35), antisense RNA (36), or selectiveinhibitors of IGF1R (37, 38)]. Downstream molecules in the IGF1Rsignaling pathway, such as AKT and mTOR, might also be atherapeutic target, as rapamycin, an mTOR inhibitor, and its esterCCI-779 show anticancer activities (39). It is not yet knownwhether any of these drugs can also modify the effect of a doubledose of IGF2 in normal cells. However, given the high prevalenceof LOI, even modest reversal of its effects could have a significant

effect on cancer prevention, similar to the use of statins to lowerlipid in averting heart disease (40).Because a limited number of patients have informative

polymorphisms to determine the LOI status of IGF2 , we mayneed better markers to predict cancer risk other than IGF2 LOIitself (e.g., target genes of IGF2 signaling or genes identified bygenome-wide expression analysis comparing LOI� and LOI+

samples). An interesting question is why a double dose of IGF2can affect cell differentiation so significantly. Insight is providedby a recent experiment in which mouse parthenogenotes werederived. Previously, this had been impossible, as the embryosform complete ovarian teratomas (i.e., disorganized relativelywell differentiated tumors containing diverse tissue types).When one maternal pronucleus was derived from the sameH19 deletion mouse strain (23) we used in our intestinalneoplasia model, fully developed mice could be obtained (41).Thus, it seems that IGF2 is a dose-dependent titrator of tissuedevelopment, consistent with the altered progenitor-differenti-ated cell balance we observed. Careful analysis of dose-dependent changes in IGF2-mediated signal transduction mayclarify this question (e.g., by observing phosphorylation andtranslocation of molecules in the IGF1R signaling pathway).Such work, in addition to its importance for cancer research,may lead to an answer to the question of why one of the twoalleles has been silenced through evolution to play a role ingrowth regulation.

Acknowledgments

Received 8/18/2005; revised 10/3/2005; accepted 10/12/2005.Grant support: NIH grant CA65145 (A.P. Feinberg).

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2005;65:11236-11240. Cancer Res   Atsushi Kaneda and Andrew P. Feinberg  Intestinal Tumor Risk

: A Common Epigenetic Modifier ofIGF2Loss of Imprinting of

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