metastasizing mammary carcinomas in h19 enhancers-igf2 transgenic mice

MAMMARY TUMORS IN Igf2 TRANSGENIC MICE 43THE JOURNAL OF EXPERIMENTAL ZOOLOGY 281:43–57 (1998)

© 1998 WILEY-LISS, INC.

Metastasizing Mammary Carcinomas in H19Enhancers-Igf2 Transgenic Mice

DIMITRINA D. PRAVTCHEVA* AND THOMAS L. WISEPediatric Research Institute, St. Louis, Missouri 63110

ABSTRACT The insulin-like growth factor II is mitogenic for a number of cell types and caninhibit apoptosis. The frequent expression of this gene in human and experimental animal tu-mors indicates that insulin-like growth factor-2 may play an important role in tumor develop-ment. It has also been hypothesized that overexpression of this growth factor may be responsiblefor the increased incidence of childhood tumors in patients with Beckwith-Wiedemann syndrome.To assess the effects of Igf2 on tumor development we produced six transgenic mouse lines thatexpress the gene under the control of the H19 enhancers. Transgenic expression was initiated inthe embryonic period but remained high in several adult tissues, including the mammary gland,lung, and liver. Adult transgenic females from five of the transgenic lines developed often mul-tiple mammary tumors that had the ability to metastasize. Increased incidence of other solidtumors was also noted in older mice. These findings indicate that Igf2 expression increases theprobability of malignant transformation and that the mammary gland is at a particularly highrisk of tumor development in response to chronic increase in Igf2 gene dosage. J. Exp. Zool.281:43�57, 1998. © 1998 Wiley-Liss, Inc.

The insulin-like growth factor II (IGFII) is a 67-amino acid peptide with mitogenic effects on anumber of cell types (Sara and Hall, ’90). It hasbeen established through targeted mutagenesis inmice that IGFII is an embryonic growth factor;mice with no active copies of the insulin-likegrowth factor-2 gene (Igf2) are 40% smaller thantheir normal littermates (DeChiara et al., ’90). Themitogenic effects of IGFII are mediated mainlythrough the type-1 IGF receptor (designated Igf1rand IGF1R in mice and humans, respectively),which also binds, with higher affinity, the insu-lin-like growth factor-I (IGFI), and, with muchlower affinity, insulin (Moxham and Jacobs, ’92;Baker et al., ’93; Liu et al., ’93; Werner andLeRoith, ’96). It was recently demonstrated thatsome of the mitogenic/growth effects of IGFII arealso mediated through the insulin receptor (IR)(Louvi et al., ’97; Morrione et al., ’97). There isanother high-affinity receptor for Igf2 (Igf2r orIGF2R), which also binds the mannose-6-phos-phate (M6P) moiety of lysosome-targeted enzymes(Kornfeld, ’92). It has been proposed that this sec-ond receptor acts as a sink for IGFII, and the phe-notype of mice that are deficient for both Igf2 andIgf2r is consistent with this idea (Filson et al.,’93; Wang et al., ’94b; Ludwig et al., ’96). The ac-tivity of IGFs is also modulated by a family ofIGF-binding proteins (IGFBPs), which are encodedby at least six different genes (McCusker and

Clemmons, ’92). The Igf2 gene is widely expressedin the embryonic period, particularly in mesoder-mal and endodermal tissues (Stylianopoulou et al.,’88b; Hedborg et al., ’94). In rodents, the serumlevels of IGFII decline after birth, whereas in hu-mans, they remain high (Daughaday and Rotwein,’89). The Igf2 gene is imprinted in both mice andhumans; it is expressed only from the paternalallele in most tissues (DeChiara et al., ’91;Giannoukakis et al., ’93; Ohlsson et al., ’93).

The IGFs act as progression factors; they arerequired for the cells to proceed from the G1 tothe S phase of the cell cycle (Werner and LeRoith,’96). They also act as antiapoptotic agents (Har-rington et al., ’94a,b; Raff et al., ’93; Resnicoff etal., ’95). The antiapoptotic effects of IGFII requirethe presence of the IGF1R, but they are manifestin stages of the cell cycle where IGFII is no longerrequired for progression (Harrington et al., ’94a,b).Both the mitogenic and the antiapoptotic effectsof IGFII can contribute to the growth autonomyand malignant phenotype of cells that express thisgrowth factor. One of the malignancies where IGFproduction has attracted much attention is breastcancer. In situ hybridization studies, RNase pro-

Grant sponsors: NIH and USDA.*Correspondence to: Dimitrina D. Pravtcheva, P.O. Box 11647, St.

Louis, MO 63105. E-mail: [email protected] 14 January 1998; Accepted 22 January 1998

44 D.D. PRAVTCHEVA AND T.L. WISE

tection assays on normal and malignant breast tis-sues, and analysis of fibroblast populations estab-lished from normal breast tissues and breast cancernodules have shown that IGFI is produced mainlyby stromal elements in normal breast tissues, butless frequently by stromal elements of breast can-cer, and in only one of the known breast cancerlines (Cullen et al., ’91, ’92; Giani et al., ’96). Onthe other hand, IGFII production has been detectedin the stromal elements of both normal and malig-nant breast tissues and is the predominant type ofIGF produced by the latter. IGFII production hasoccasionally been detected within the epithelialcomponent of the tumors as well, and IGFII is mi-togenic for breast tumor epithelial cells (Karey andSirbasku, ’88; Stewart et al., ’90; Giani et al., ’96).

IGF1R is also expressed in freshly removed nor-mal and malignant breast tissues and in mostbreast tumor cell lines. These findings support theidea that IGFII is involved in the control of breastepithelial growth through paracrine effects (Cullenet al., ’91, ’92; Giani et al., ’96). Blockage of theIGF1R with a specific antibody resulted in sup-pression of tumor xenograft growth, and growthof tumor cells in culture, again suggesting the im-portance of IGFs for the growth of these tumors(Rohlik et al., ’87; Pekonen et al., ’88; Arteaga andOsborne, ’89; Peyrat and Bonneterre, ’92).

Additional support for the idea of IGFII involve-ment in the control of tumor cell proliferation comesfrom findings in patients with Beckwith-Wiede-mann syndrome (BWS). BWS is associated withfetal overgrowth and a high incidence of severalchildhood tumors: nephroblastoma (Wilms tumor),hepatoblastoma, rhabdomyosarcoma, adrenocorti-cal carcinoma, and pancreatic carcinoma (Beckwith,’69; Wiedemann, ’83; Junien, ’92). Because of itschromosome position, imprinting pattern, and mi-togenic effects, it is believed that the IGF2 genealone, or in conjunction with other genes in thisregion (Thompson et al., ’96; Hatada et al., ’96),may play a role in the development of the BWS.The tumors associated with BWS show increasedlevels of IGF2 transcripts (Hedborg et al., ’94; Reeveet al., ’85; Scott et al., ’85; Haselbacher et al., ’87).The organs that most frequently develop neoplasmsare the same as the organs that show a high levelexpression of IGF2 (Hedborg et al., ’94) and asym-metrical growth. High levels of IGFII have alsobeen found in spontaneous or experimentally in-duced tumors of the same organs (Cariani et al.,’88, ’91; Schirmacher et al., ’92).

These data support the notion that increasedlevels of IGFII may be associated with an in-

creased risk of tumor development. A logical wayto test this hypothesis is by creating Igf2 trans-genic mice. Several laboratories have reported theproduction of transgenic mice that overexpressIgf2 under the control of heterologous promotersactive in late fetal or postnatal life (Rogler et al.,’94; van Buul-Offers et al., ’95; Ward et al., ’94).Increased incidence of tumors was reported by twogroups. Mice that overexpressed IGF2 under thecontrol of the major urinary protein (MUP) pro-moter developed tumors in a variety of organs(most often hepatocellular carcinoma and lym-phoma) after the age of 18 months (Rogler et al.,’94). Directing Igf2 expression to the mammarygland with a sheep β-lactoglobulin promoter re-sulted in an increased incidence of mammary tu-mors (Bates et al., ’95), although no metastaseswere reported in these mice.

Embryonic overexpression of IGFII has beenachieved in mice with a maternally inherited H19knock-out (KO) allele. The H19 gene is situated~80–90 kb 3´ of Igf2, and is expressed only fromthe maternal allele (Bartolomei et al., ’91; Zemelet al., ’92; Zhang et al., ’93). It has been proposedthat the Igf2 and H19 genes are regulated by com-mon enhancers, because the two genes are neigh-bors, have similar patterns of expression duringdevelopment, and have opposite imprinting. Accord-ing to this model, the access of Igf2 to these en-hancers is determined by the imprinting of H19(Bartolomei et al., ’92). A set of enhancers with en-dodermal specificity is located in the 3´ flank ofthe H19 gene (Yoo-Warren et al., ’88) (the meso-dermal enhancers have not yet been identified). Inthis enhancer competition model, the active H19gene on the maternal chromosome precludes high-level expression from the maternal Igf2 allele,whereas the silence of the paternal H19 gene al-lows the paternal Igf2 gene to utilize these enhanc-ers. The phenotype of mice that have an inactiveH19 gene or inactive endodermal H19 enhancersis in agreement with the predictions of this model.Inheritance of the H19 KO allele through themother resulted in activation of the maternal Igf2allele, and an increase in Igf2 steady-state mRNAlevels that was most pronounced in mesodermaltissues (Leighton et al., ’95a; Ripoche et al., ’97).These mice grew to a larger size than their nor-mal littermates, but so far have not shown an in-creased incidence of tumors (Leighton et al., ’95a,b).An increase of IGFII levels in the embryonic pe-riod was also observed in mice with inactive Igf2rgenes. These mice are also larger than normal, butdie in late fetal or early postnatal life (Johnson,

MAMMARY TUMORS IN Igf2 TRANSGENIC MICE 45

’74; Winking and Silver, ’84; Lau et al., ’94; Wanget al., ’94b; Ludwig et al., ’96).

We have produced Igf2 transgenic mice that ex-press the gene in the embryonic period under thecontrol of the H19 enhancers (Wise and Pravt-cheva, ’97). Our goal in producing these mice wasto assess the effect of an increased dosage of IGFII(in the period when the gene is normally active)on the growth and tumor incidence of the trans-genic animals, without altering the activity ofother genes. We did not observe an increase inthe incidence of tumors in young mice that canbe considered the equivalent of the tumors foundin BWS patients. Adult transgenic mice, however,continued to express the gene in several organs,including the mammary gland, lung, and liver.Transgenic females developed often multiple mam-mary tumors, that had the ability to metastasize.Increased incidence of lung tumors was also notedin older transgenic mice. These results indicatethat chronically elevated levels of Igf2 expressionincrease the risk of malignant transformation andthat the mammary gland is at a particularly highrisk of tumor development as a result of an in-crease in Igf2 dosage.

MATERIALS AND METHODSEmbryo microinjection

All embryo manipulations were performed asdescribed (Pravtcheva et al., ’91). FVB/N one-cellembryos were used for the microinjections. In-jected embryos were transferred into fosters onthe same day, or after overnight culture. In thelatter case, only embryos that had divided wereused for the transfer. (C57Bl/6J × DBA/2)F1(BDF1) or CD-1 females mated with vasectomizedBDF1 males were used as fosters. The transgeniclines were maintained on an FVB/N backgroundby crosses with FVB/N mice, or by crosses betweentransgenic mice. Female transgenic mice werekept in the colony until they developed tumors ordied of unrelated causes.

Construct preparationThe construction of the H19eIgfMlu clone was

described in detail elsewhere (Wise and Pravt-cheva, ’97). The injected construct (Fig. 1) containsa 17-kb EcoRI fragment of cosIGF4 (Rotwein andHall, ’90), including the entire coding region ofIgf2. A small 5´ fragment of the non-coding exon1 was left out in this construct. The MluI site atthe 3´ end of the nontranslated portion of exon 6was abolished to allow the distinction of the

transgene DNA or transcripts from the endogenousgene after PCR amplification and digestion withMluI. An approximately 7-kb genomic fragmentcontaining the H19 enhancers was placed at the5´ end of the EcoRI fragment, so that the enhanc-ers are proximal to the Igf2 gene. Some of thefemale mice used as controls for evaluating mam-mary tumor incidence contained one of two differ-ent constructs (designated as B and C on Fig. 1).Both of these constructs contain the entire genomicregion present in cosIGF4 (Rotwein and Hall, ’90)except for the replacement of the coding portion ofthe Igf2 gene (exons 4, 5, and the 5´ portion of exon6) with the lacZ reporter. Construct C contains theH19 enhancers (as part of the 7 kb HindIII frag-ment) at its 3´ end. Neither of these constructs iscapable of producing IGFII. A more detailed descrip-tion of the construction and expression pattern ofthese clones will be presented elsewhere. For em-bryo microinjections, the genomic insert of H192-IgfMlu was separated from plasmid sequences bydigestion with NotI and ClaI, followed by sucrose-gradient centrifugation or agarose-gel electrophore-sis and purification by passing through an Elutipcolumn (Schleicher and Schuell, Keene, NH).

Genotyping of transgenic progenyDNA was extracted from tails or spleens (Sam-

brook et al., ’89; Hogan et al., ’94). Transgenicfounders and progeny were identified by South-ern analysis, using probes from the Igf2 gene orthe H19 enhancers, and/or by PCR (Wise andPravtcheva, ’97). For Southern analysis, 10 µg ofDNA were digested with restriction enzymes un-der conditions recommended by the manufacturer.The DNA was run on 0.6–0.7% agarose gels, blot-ted, and hybridized with probes labeled by the ran-dom-primer method. For PCR analysis, a 393-bpfragment of exon 6 including the MluI (or modi-fied MluI) site was amplified using the followingprimers: 5´-TTGGAGACTATGAATTGGCCT-3´, 5´-AATGCATGTTAGAGGGACTGA-3´. Amplifica-tions were done at 95°C, 1 min, 55°C, 1 min, 72°C,2 min, 40 cycles, using BRL Taq polymerase andreaction buffer. Digestion of the amplificationproducts with MluI cleaves the endogenous Igf2product into two fragments (298 bp and 95 bp),whereas the amplification product of the trans-gene is resistant to MluI digestion.

Expression analysis of the transgenesTransgene expression was monitored by RT-

PCR, using the same set of primers bracketingthe MluI (or the abolished MluI) site. RNA was


purified from tissues according to the method ofChomczynski and Sacchi (’87) and digested withRNase-free DNase I prior to the amplification. Forthe reverse transcriptase reaction, 250 ng RNAwas first heated at 70°C for 10 min. and cooledon ice. The RNA was then incubated for 1 h at45°C in 200 u Superscript II reverse transcriptase(BRL), 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3.0mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTPs,and 0.75 µM 3´ “MluI” primer (20 µl total vol-ume). For the PCR reaction, the 5´ “MluI” primer,Taq polymerase, and the appropriate amount of10× PCR reaction buffer and MgCl2 were addedto the reverse transcriptase reaction (100 µl totalvolume) and amplified as described above. Actinprimers (5´-GCCGGGACCTGACGGACTAC, 5´-GGGGCCCGGACTCATCGTACT) were used ascontrols for loading.

Preparation of mammary glandwhole mounts

Mammary gland whole mounts were preparedas described by Sympson et al. (’94). In all casesthe right abdominal mammary gland was dis-sected out and spread on a glass slide. The glandwas fixed overnight in Carnoy’s fixative (75% etha-nol, 25% acetic acid), treated briefly with 70% etha-nol, and stained overnight in carmine alum (0.2%carmine dye, 0.5% aluminum potassium sulfate).The glands were dehydrated by passing through aseries of 50%, 70%, 95%, 100%, and 100% ethanol(1 h each), defatted by overnight treatment withtoluene, and stored in methyl salicylate.

RESULTSWe have produced eight transgenic founders

(pmH19eIgfMlu Fos 1, 10, 16, 18, and 22 andumH19eIgfMlu Fos 2, 4, and 14) with the H19eIgf-Mlu construct. The production of these lines, theexpression of the transgene in the embryonic pe-riod, and its effect on the viability of newborn ho-mozygous transgenic mice were described in detailin a separate report (Wise and Pravtcheva, ’97).Founder 16 died for unknown reasons and Fo 22

was stillborn. We have established lines 1A, 1B,10A, 10B, 18A, and 2 from the correspondingfounders. The mosaic founders 4 and 14 producedoffspring that died around the time of birth (seebelow). A and B lines originate from commonfounders but differ in their restriction pattern andmay represent independent transgene integrationsor rearrangements of the original transgene lo-cus. The transgene was found to be expressed inthe embryonic period in liver, lung (with a tran-script level 1 to 2.7 times that of the endogenousgene), kidney, intestine, and brain (at 0.5 to 1times the level of the endogenous gene), and at alower level in heart, muscle and skin. Heterozygousday 17–19 embryos showed a small difference insize compared with nontransgenic littermates. Allhomozygous mice from lines 1A and 2 and most ofthe homozygous mice of line 10A died around thetime of birth; so did nonmosaic heterozygous prog-eny of founders umH192IgfMlu 4 and 14 (Wiseand Pravtcheva, ’97). Crosses of heterozygous par-ents of line 10B also produced stillborn progeny,although the number of mice from this line wehave analyzed is smaller. Heterozygous progenyof lines 1A, 1B, 10A, 10B, 18A, and 2 are viableand show few obvious differences at weaning com-pared to their nontransgenic littermates (see be-low). Because of the lower level of expression ofthe transgene in line 18A, these mice have notbeen analyzed in detail.

Transgene expression in adult H19-enhancers Igf2 transgenic mice

Previous reports have indicated that transcrip-tion of the rodent Igf2 gene declines rapidly afterbirth in most tissues except the choroid plexusand the leptomeninges (Stylianopoulou et al.,’88a,b; Lee et al., ’90). We analyzed transgene ex-pression in adult transgenic females from linespmH19eIgfMlu 1A (age 8 months), pmH19eIgMlu10A (age 16 months), and umH19eIgfMlu 2 (age7 months) by RT-PCR (data for lines 1A and 10Ashown in Fig. 2). The RT-PCR product of thetransgene transcript was distinguished from thatof the endogenous transcript by its resistance tocleavage by MluI. Transgene expression at vary-ing levels was found in most of the analyzed or-gans (liver, kidney, lung, heart, and mammarytissue). With this assay, we also detected expres-sion of the endogenous Igf2 gene in kidney andheart. Although this assay is not strictly quanti-tative and tends to exaggerate the amount of thetransgene transcript, the data presented in Fig-ure 2 support the conclusion that in all of the

Fig. 1. Structure of construct H19eIgfMlu, used for micro-injection. The organization of the Igf2 gene and its splicingpattern are indicated on the top. P1, P2, and P3 designatepromoters 1, 2, and 3, respectively. Numbers 1–6 designateindividual exons. Coding portions are shown in black. P1 anda 5´ segment of exon 1 are not included in this construct. ∆MluImarks the position of the abolished MluI site. Horizontal ar-rows mark the position of primers used for PCR and RT-PCR.In constructs B and C the entire coding portion of Igf2 is re-placed by the lacZ reporter. E = EcoRI; N = NruI; Z = ZhoI.


tissues analyzed, except kidney, transgene ex-pression was as high or higher than that of theendogenous gene. We detected low levels of en-

dogenous Igf2 transcripts in mammary tissue oftransgenic (Fig. 2A) and nontransgenic females(Fig. 2C). (Note that the relatively strong Igf2 sig-nal of the nontransgenic mammary gland in Fig-ure 2C is mirrored by a much stronger signal inthe actin RT-PCR control from the same tissue,and thus cannot be viewed as a quantitative indi-cator of the level of endogenous Igf2 expression.)Our results indicate that the rodent Igf2 gene con-tinues to be transcribed (although at a low level)in some tissues other than the choroid plexus andthe leptomeninges after birth.

Adult transgenic mice showed few obvious dif-ferences compared with their nontransgenic lit-termates (including differences in size). The mostconsistent abnormality we observed in these micewas a variable degree of teeth enlargement. Theteeth abnormalities in the heterozygous transgenicmice and the frequently observed cleft palate inour homozygous mice (Wise and Pravtcheva, ’97)may share a common developmental cause.

Heterozygous H19 enhancers-Igf2 transgenicmice develop multiple mammary tumors

that can metastasizeFemale mice of transgenic lines 1A, 1B, 10A,

10B, and 2 develop mammary tumors (Table 1,data for line 10B not included). The vast majorityof these tumors were found in females that hadhad at least one litter and were older than 6months (the youngst age being 5.5 months).Eighty-one percent of parous females (older than6 months) of line 1A, 50% of females of line 1B,64% of females of line 10A, and 100% of femalesof line 2 developed mammary tumors (Table 1),with a mean age at tumor discovery of 9–10months. We have also observed tumors in femalesfrom line 10B, but the number of females fromthis line at the susceptible age is small. Six ofnine transgenic females (two each from lines 1A,10A, and 2) that had no recorded litters also de-veloped tumors, with a mean age at tumor dis-covery of 13.4 months. These females, however,had been caged with males for at least some pe-riod of time, and may have had small litters thatwere cannibalized soon after delivery and werenever discovered. The tumors consisted of moder-ately to poorly differentiated cells arranged in tu-bules or acini, and showed aggressive, infiltrativegrowth. Morphologically, the majority of the tu-mors resembled adenocarcinomas type A or typeB (Squartini and Pingitore, ’94) (Fig. 4). Some tu-mors showed characteristics of adenoacanthomasor adenocarcinomas with squamous components.

Fig. 2. RT-PCR analysis of transgene expression in adulttissues. (A) Tissues from line pmH19eIgfMlu 1A. (B) Tissuesfrom line H19eIgfMlu 2. (C) Tissues from nontransgenic FVB/N mice. The larger fragment resistant to cleavage by MluI isderived from the transgene. Actin RT-PCR controls are shownbelow each “MluI” Igf2 RT-PCR.


Two-thirds (65%) of the affected females developedmultiple tumors (Fig. 3). This number is likely tobe an underestimate, because upon excision of alarge tumor, small additional tumors were not al-ways sought. If left to grow on the host, the tu-mors metastasized, with involvement of thespleen, liver, and lung (Figs. 4C,D). Macroscopi-cally visible metastases were found in 13 females.The mammary tumor origin of the metastatic nod-ules was confirmed by histological analysis (Figs.4C,D). The spleen was involved most frequently(eight cases), followed by lung (six cases), and liver(two cases). The control group included FVB/Nmice and Igf2-lacZ transgenic mice (Table 2). TheIgf2-lacZ transgenes have the entire coding por-tion of Igf2 replaced by the lacZ reporter (con-structs B and C in Fig. 1; Wise and Pravtcheva,unpubl.), and thus are incapable of producingIGFII. The genetic background of the Igf2-lacZtransgenic mice is also FVB/N. In the controlgroup (Table 2), a single mammary tumor was ob-served in an 18.5-month-old female mouse that con-tained an Igf2-lacZ construct (a frequency of 2%).The mean age of the females in the control groupwas higher than the mean age at tumor discoveryof mice in the experimental group, and all of thecontrol females had had multiple litters.

RT-PCR analysis of normal and malignantbreast tissue from lines 1A (mice 1–13, 1–15, 1–17, 1–38, 1–41), 1B (mouse 1–3) (Fig. 5), 10A, and2 (not shown) revealed the predominant presenceof the transgene-derived transcript. In the laterstages of PCR amplification, heteroduplexes formbetween DNA strands that contain the MluI site(RT-PCR products of the endogenous Igf2 gene)and strands that lack the MluI site (RT-PCR prod-ucts of the transgene). Because these heterodu-plexes are resistant to MluI cleavage, the strongerexpression of the transgene tends to mask the lowlevel expression from the endogenous gene. Theweak endogenous band, seen in normal transgenicmammary tissue in Figure 2A, is usually not vis-ible in the tumors by this assay, but can be de-tected in some tumors with a more sensitive PCRassay that uses radioactive label.

We examined whole mounts of mammary glandsfrom 18 transgenic females (from lines 1A, 1B,10A, 10B, and 2) and 11 controls (FVB/N femalesor nontransgenic littermates), for the occurrenceof focal hyperplastic lesions resembling hyperplas-tic alveolar nodules, or HANs (Medina, ’73;Squartini and Pingitore, ’94) that precede the de-velopment of MMTV-induced mammary tumors.All of the examined females were adults, with a

TABLE 1. Mammary tumor incidence in H19eIgfMlu transgenic parous females

Total no. of parous No. of mice Mean age at tumorMouse line females >6 months old with tumors (%) discovery (months)

pmH19eIgfMlu 1A 21 17 (81%) 8.9pmH19eIgfMlu 1B 4 2 (50%) 10.0pmH19eIgfMlu 10A 11 7 (64%) 8.9*umH19eIgfMlu 2 8 8 (100%) 9.4*The founder mouse, which was a mosaic, was excluded from this calculation.

Fig. 3. Multiple mammary tumors in H19eIgfMlu trans-genic mice. The mammary glands in the mouse branch outin the subcutaneous fat pads, which are wrapped around

the body wall, accounting for the dorsal position of some ofthe tumors. (Left) Mouse from line pmH19eIgfMlu 1B. (Right)Mouse from line umH19eIgfMlu 2.


range of ages from 9.5 to 18 months for thetransgenic females, and 9.5 to 17 months for thecontrols. The morphology of the mammary glandsin the examined females showed considerablevariation depending on the age of the mice andthe occurrence of previous pregnancies. Some ofthe transgenic females had tumors in their othermammary glands. Foci of abnormal growth werediscovered in nine of the transgenic females (Fig.6). The number of these lesions (0–4/gland)roughly corresponded to the number of separatetumor nodules that could be found in an individualmammary gland of tumor-bearing females. Al-though the lesions found in whole mounts havenot been sectioned, they most likely correspond

to the small macroscopically visible nodules thatcould be found as singles, or in low numbers, insome transgenic mammary glands. These noduleshave the histological appearance of adenomas (Fig.6B). In addition, we have examined on wholemounts the morphology of the mammary glandsof four younger (age 4–8 months) parous trans-genic females (one from line pmH19IgfMlu 1A andthree from line umH19IgfMlu 2) and four non-transgenic littermates or age-matched FVB/N con-trols with a similar reproductive history (numberand timing of litters). No hyperplastic alveolarnodules were noted in these mice. More subtle ortransient changes in mammary gland morphol-ogy cannot be ruled out by these observations.

Fig. 4. Histology of mammary tumors in H19eIgfMlutransgenic mice. (A) Section of a mammary tumor inpmH19eIgfMlu Fo 1. The tumor was moderately cellular,multilobular, and infiltrative. The cells were arranged in lob-ules or acini, lined by three to five layers of cells and fre-quently containing proteinaceous material and cell debris. Thestroma was fibrous and delicate. Cells were large, closelypacked, with eosinophilic to slightly basophilic cytoplasm, andprominent nucleoli. The tumor was classified as adenocarci-

noma type B. (B) A mammary tumor in an F1 female fromline pmH19eIgfMlu 1B. This tumor contained smaller cells,with less prominent nucleoli, that formed smaller, more regu-lar glands lined by one to three layers of cells. There wasmore significant stromal involvement. This tumor resembleda type A adenocarcinoma. (C) Metastasis of the mammarytumor in Fo 1 in the liver. (D) Metastasis of the mammarytumor in Fo 1 in the spleen.


We have examined 19 older (mean age 19.5months) Igf2 transgenic mice for the occurrenceof tumors in other organs. The mice in this groupwere dissected at the time of their removal fromthe colony because of old age without any otherselection (e.g., they did not look obviously sick atthe time). The majority of the mice in this groupare male (18 males, 1 female), since most femaleIgf2 transgenic mice that had had at least onelitter had succumbed to mammary tumors beforethis age. Nine of these mice (or 47%) had devel-oped tumors. The most frequent location of thetumors was the lung (eight cases, 42%); there was,in addition, one case of lipoma. Only organs thathad macroscopically visible lesions were subjectedto histopathological examination and thus smallerbenign or early malignant lesions could have beenmissed. The control group consisted of 10 mice of

a similar age (mean age 20.4 months) and sex com-position (nine males and one female), which con-tained two different Igf2-lacZ constructs (B and C).In both constructs, the entire coding portion of theIgf2 gene is excised and replaced by the lacZ re-porter, and the genetic background of these mice isalso FVB/N. One of the mice in this group (10%)had a lung tumor. The frequency of lung tumors inour control mice was similar to the incidence of al-veolar-bronchiolar lung carcinomas in older FVB/Nmice (Mahler et al., ’96): 7% and 5% in 14-month-old males and females, and 14% and 13% in 24-month-old males and females, respectively (Mahleret al., ’96). No mammary tumors were reported inthe Mahler et al. (’96) study. Our findings indicatethat chronically elevated local levels of Igf2 expres-sion can lead to an increased frequency of tumorsin the lung, that develop with a long latency.

TABLE 2. Mammary tumor incidence in control parous females

Total no. of Age at tumorparous females Age range in No. of mice with discovery

Mouse line >6 months old months (mean) tumors (%) (months)

FVB/N 23 0Igf2-lacZTransgenic females* 21 1Total 44 6.5–19 (11.8) 1 (2%) 18.5*Transgenic females produced with two different Igf2-lacZ constructs, which do not contain the coding portion of the gene.

Fig. 5. RT-PCR analysis of transgene expression in mam-mary tumors and normal mammary tissue of H19eIgfMlutransgenic mice. The numbers above the lanes identify the host.The two lanes with number 1-17 contain RNA from two sepa-

rate tumors in the same host. Lane 10-77 contains RNA froma thymus sarcoma in male 10-77. A hyperplastic lymph nodefrom mouse 1-38 (line pmH19eIgfMlu 1A) is also included.


DISCUSSIONWe have produced transgenic mice that express

the Igf2 gene under the control of the H19 en-hancers and have examined these mice for the fre-quency and type of tumor development. The H19enhancers, which have predominantly endodermalspecificity, directed high-level expression in theembryonic lung and liver, and a more moderateincrease in expression in intestine. Expression insome nonendodermal tissues was also noted (Wiseand Pravtcheva, ’97). The transgene continued tobe expressed in the postnatal period in severalorgans, including the lung and liver, and was alsoexpressed in the mammary gland.

Female mice from five of our transgenic linesdevelop mammary tumors with a high incidence(Fig. 3, Table 1); the absence of tumors in the sixthline (18A) was most probably due to the lowerlevel of expression of the transgene in this line.The mean age of tumor appearance was compa-rable to that of mice expressing an MMTV-myctransgene (Stewart et al., ’84), and considerablyyounger than the MMTV-cyclin D1 transgenicmice (Wang et al., ’94a). The tumors in the H19enhancers-Igf2 transgenic mice are among a mi-nority of described transgene-induced mammarytumors that have the ability to metastasize (re-viewed in Amundadottir et al., ’96). All of the tu-mors we have analyzed so far express the Igf2transgene (Fig. 4), as does normal mammary tis-sue from these transgenic females (Figs. 2, 4). Itis interesting to note that Fo 10, which was a mo-saic and developed a mammary tumor at an ad-

vanced age (20 months), showed the lowest levelof transgene expression among all the tumors wehave analyzed. Because of the very low incidenceof spontaneous mammary tumors in FVB/N mice,and their appearance at a more advanced age(Table 2; Wang et al., ’94), we conclude that thehigh mammary tumor incidence in the Igf2 trans-genic mice is due to the expression of the Igf2transgene. The development of mammary tumorsin five transgenic lines, three of which were de-rived from independent founders, indicates thattransgene expression in the mammary gland isnot a result of a position effect. Because no mam-mary tumors were reported in several other Igf2transgenic lines with very high serum IGFII lev-els, and where expression was targeted to organsother than the mammary gland, we conclude thatthe tumors in our transgenic lines are due to thelocal autocrine or paracrine effects of Igf2 in themammary gland. The low level of endogenous Igf2transcripts in the mammary gland suggests thata negative regulatory element that normally sup-presses Igf2 activity in this tissue is absent fromour construct. The rodent Igf2 gene is known tobe activated in some chemically induced mam-mary tumors, for example, in DMBA-induced tu-mors in the rat (Manni et al., ’94). Igf2 is alsoexpressed in some mouse mammary tumor lines,this expression is associated with a higher meta-static potential (Guerra et al., ’96). As mentionedpreviously, mammary tumors also develop intransgenic mice expressing Igf2 under the controlof a sheep β-lactoglobulin promoter (Bates et al.,

Fig. 6. Isolated foci of pre-neoplastic or early neoplasticgrowth in the mammary glands of H19 enhancers-Igf2transgenic mice. (A) Whole mount mammary gland prepara-tion from a pm H19eIgfMlu 10A female. The foci of abnormalgrowth are visible as the more darkly stained formations and

are indicated by arrowheads. (B) Section of a small nodule inthe mammary gland of a female from line 1A. The noduleshowed hyperplasia and intralobular fibrosis without evidencefor cell atypia and was classified as an early adenoma.


’95). This promoter is active only during preg-nancy and lactation and thus would affect mam-mary epithelium growth and differentiation, orprogrammed cell death, during these periods. Incontrast, the H19 enhancers-Igf2 transgene is con-tinuously active in the adult mammary gland.This expression pattern more closely approximatesthe situation in humans, where deregulation ofIgf2 expression in the stroma may be an early,pre-cancer stage event (see below), which persistsoutside of the cycles of pregnancy and lactation.The tumors in β-lactoglobulin-Igf2 transgenic micewere found to be transplantable, although no me-tastases were reported (Bates et al., ’95). The me-tastases that developed in our transgenic miceshowed an unusual preference for the spleen(whereas lung is the most common site observedin other mouse mammary tumor models). LocalIGFII levels are unlikely to be the determiningfactor for the frequent development of metastasesin the spleen (Fig. 2).

Our results indicate that increased gene dos-age of Igf2 (presumably associated with higher lev-els of IGFII) in the normal mammary glandstrongly increases the probability of epithelialmalignant transformation. At present we do notknow what cell type in the mammary gland ex-presses the Igf2 transgene. The mechanism of thiseffect is also not understood. Given the mitogenicand antiapoptotic effects of IGFII one might ex-pect to find diffuse, or multifocal hyperplasia inthe premalignant mammary gland of our trans-genic mice. Multiple HANs precede the develop-ment of mammary tumors induced by MMTV(Medina, ’73; Squartini and Pingitore, ’94) orTGFα overexpression (Sandgren et al., ’95). Mul-tiple pre-neoplastic lesions have also been observedin a well-studied animal model of multistage tu-morigenesis with Igf2 reactivation—the develop-ment of pancreatic carcinomas in RipTag mice(Naik et al., ’96). So far, however, we have foundno evidence for such changes in our mice. The hy-perplastic lesions of the type illustrated in Fig-ure 5 were usually solitary or present in a lownumber per gland (and were altogether absentfrom many of the glands). Absence of manifestpreneoplastic hyperplasia was also noted inMMTV/c-myc and MMTV/v-Ha-ras transgenicmice, where the mammary tumors were observedto arise from morphologically normal epithelium(Leder et al., ’86; Sinn et al., ’87). An alternativemechanism for the tumorigenic effect of IGFII maybe through inhibition of apoptosis in cells thathave suffered genetic damage. This inhibition

would allow the accumulation of genetic changesin these cells that ultimately lead to malignancy(Harrington et al., ’94a,b). We are currently ex-amining the Igf2 transgenic mice for more subtleor transient alterations in their mammary glandmorphology.

IGF2 expression by stromal elements has beennoted in a high proportion of human breast tu-mors. IGF1 expression, on the other hand, ismostly found in the stromal elements of normalbreast tissue or benign lesions, but in a lower pro-portion of breast tumor stroma (Cullen et al., ’91,’92; Giani et al., ’96; Quinn et al., ’96). Additionalevidence for a possible role of IGF2 in tumor de-velopment in humans comes from several recentreports of frequent loss of heterozygosity for theIGF2R gene, and mutations in the remaininggene, in tumors of the liver and breast (De Souzaet al., ’95; Hankins et al., ’96). These findings sug-gest that this receptor acts as a tumor suppres-sor. At least some of the antitumor effects of theIGF2R may be due to its ability to mediate IGFIIdegradation. Another mechanism that has the po-tential to increase local IGFII levels is loss of im-printing (LOI), resulting in reactivation of thematernal IGF2 allele. IGF2 LOI has been seen insome human breast carcinomas and premalignantlesions (McCann et al., ’96; Yballe et al., ’96). Thefindings of LOI in premalignant lesions imply arole of IGFII in the early stages of breast tumordevelopment. In vitro co-culture experiments havesuggested that mammary tumor epithelium in-duces IGF2 expression by the surrounding stro-mal elements (Singer et al., ’95). These findingsindicate that IGF2 expression in the mammarygland may continue to play a role in tumorigen-esis even after the establishment of fully malig-nant epithelial cell clones. Transgenic mousemodels of mammary tumorigenesis under the in-fluence of Igf2 will facilitate the analysis of therole of this growth factor in different stages of theneoplastic process.

As mentioned previously, the H19 KO mice donot develop tumors (Leighton et al., ’95a,b). In theH19 KO mice, the regulatory elements requiredfor the postnatal down-regulation of Igf2 expres-sion are presumably intact, while, in the H19enhancers-Igf2 construct, these elements are ap-parently missing. The resulting difference in thepostnatal levels of Igf2 expression probably ac-counts for the difference in tumor incidence be-tween the H19 KO and the H19 enhancers-Igf2transgenic mice.

It is believed that the mitogenic and anti-


apoptotic effects of IGFII are mediated mainlythrough the type 1 IGF receptor (Harrington etal., ’94a,b; Ellis et al., ’96; Werner and LeRoith,’96). This receptor also binds (with severalfoldhigher affinity) IGFI. It is therefore interestingthat overexpression of IGFI in the mammarygland, driven by the whey acidic protein promoter,was associated with delayed involution after lac-tation on some genetic backgrounds, but few(Hadsell et al., ’96) or no (Neuenschwander et al.,’96) mammary tumors were reported. No increasein tumor incidence has been reported in anotherIGF1 transgenic model, where IGF1 was over-expressed under the control of the metallothionein1 promoter (Mathews et al., ’88). It is possible thatthe different findings in IGF1 and Igf2 transgenicmice are a consequence of using different promot-ers. However, these findings are consistent withthe observation that IGF2 is more frequently ex-pressed in stroma associated with tumor epithe-lium in human breast tumors, and suggest thatIGFII may be more effective than IGFI in initiat-ing or promoting mammary tumor growth. Thepresence of a second receptor (IR) mediating IGFIIaction may give this growth factor an advantageover IGFI. It is also possible that the IGFs influ-ence other functions of the proteins to which theybind. For example, IGF2R also binds (through adifferent site) proteins that are tagged with M6Presidues (Kornfeld, ’92). There are some reportsindicating that binding of IGFII (in particular thelarger precursor forms of IGFII) with IGF2R mayinterfere with binding of M6P-tagged proteins tothe receptor (Kiess et al., ’89; Mathieu et al., ’90;De Leon et al., ’95). Among this latter group ofproteins is the lysosomal enzyme cathepsin D,whose misrouting may increase extracellular ma-trix degradation and increase the metastatic po-tential of breast cancer cells (Mathieu et al., ’90;De Leon et al., ’95). Another group of proteinsbound by the IGF2R are the TGFβs, whose con-version from latent into mature form requiresbinding to this receptor (Dennis and Rifkin, ’91).The TGFβs are potent inhibitors of epithelial cellgrowth, play an important role in normal mam-mary gland development (Robinson et al., ’91), andhave a protective effect with regard to mammarytumor development in transgenic mice (Pierce etal., ’95). Interestingly, TGFβ2-induced growth in-hibition of a mammary carcinoma cell line appearsto be mediated by IGFBP3, and growth inhibi-tion was significantly reduced in the presence ofIGFII, as a result of interference with IGFBP3binding to its own receptor (Oh et al., ’93b, ’95;

Gucev et al., ’96). Moreover, IGFII was reportedto have a higher binding affinity than IGFI forIGFBP3 (Oh et al., ’93a). An overabundance ofIGFII thus may interfere with more than onegrowth regulatory pathway in the mammarygland. A more detailed analysis of the similari-ties and differences in the effects of IGFI andIGFII in the mammary gland is likely to revealimportant clues to the mechanism by which Igf2induces mammary tumor development.

ACKNOWLEDGMENTSWe thank Dr. Peter Rotwein for the mouse Igf2

genomic clone cos IGF4 and Dr. Marie La Reginafor histopathological examination of the tumorsamples. We also thank Sara Seematter for excel-lent technical assistance. This work was supportedby grants from NIH and the USDA to D.P. Earlystages of this work were supported by the CardinalGlennon Children’s Hospital New Project Develop-ment Fund, by a Cancer Research InstitutionalCommittee Award, and by the Morrissey Fund.

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