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[Cancer Research 66, 1964-1973, February 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Dominant-Stable ß-Catenin Expression Causes Cell Fate Alterations and Wnt Signaling Antagonist Expression in a Murine Granulosa Cell Tumor Model

Derek Boerboom1, Lisa D. White2, Sophie Dalle3, José Courty3 and JoAnne S. Richards1

Departments of 1 Molecular and Cellular Biology and 2 Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas and 3 Laboratoire de Recherche sur la Croissance, la Regénération et la Réparation Tissulaires, Universite Paris XII-Val de Marne, UMR Centre National de la Recherche Scientifique 7149, Creteil, France

Requests for reprints: JoAnne Richards, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: 713-798-6259; E-mail: joanner{at}bcm.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Wnt/ß-catenin signaling is normally involved in embryonic development and tissue homeostasis, and its misregulation leads to several forms of cancer. We have reported that misregulated Wnt/ß-catenin signaling occurs in ovarian granulosa cell tumors (GCT) and have created the Catnbflox(ex3)/+;Amhr2cre/+ mouse model, which expresses a dominant-stable mutant of ß-catenin in granulosa cells and develops late-onset GCT. To study the mechanisms leading to GCT development, gene expression analysis was done using microarrays comparing Catnbflox(ex3)/+;Amhr2cre/+ ovaries bearing pretumoral lesions with control ovaries. Overexpressed genes identified in Catnbflox(ex3)/+;Amhr2cre/+ ovaries included the Wnt/ß-catenin signaling antagonists Wif1, Nkd1, Dkk4, and Axin2, consistent with the induction of negative feedback loops that counteract uncontrolled Wnt/ß-catenin signaling. Expression of the antagonists was localized to cells forming the pretumoral lesions but not to normal granulosa cells. Microarray analyses also revealed the ectopic expression of bone markers, including Ibsp, Cdkn1c, Bmp4, and Tnfrsf11b, as well as neuronal/neurosecretory cell markers, such as Cck, Amph, Pitx1, and Sp5. Increased expression of the gene encoding the cytokine pleiotrophin was also found in Catnbflox(ex3)/+;Amhr2cre/+ ovaries and GCT but was not associated with increased serum pleiotrophin levels. In situ hybridization analyses using GCT from Catnbflox(ex3)/+;Amhr2cre/+ mice revealed that Wnt/ß-catenin antagonists and neuronal markers localized to a particular cell population, whereas the bone markers localized to a distinct cell type associated with areas of osseous metaplasia. Together, these results suggest that misregulated Wnt/ß-catenin signaling alters the fate of granulosa cells and that the GCT that arise in Catnbflox(ex3)/+;Amhr2cre/+ mice result from the clonal expansion of metaplastic cells. (Cancer Res 2006; 66(4): 1964-73)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Wnts comprise a large family of secreted, local-acting signaling glycoproteins known mostly for the critical roles they play in embryonic development (1, 2). Following embryogenesis, Wnts and Wnt signaling pathway components continue to be expressed in many tissues, leading to the widely held view that Wnts play additional roles in adult tissue homeostasis (3, 4), although the precise nature of these homeostatic roles remains poorly defined. Recently, several lines of evidence have converged to suggest that Wnt signaling is required for the maintenance or proliferation of stem/progenitor cell types in several tissues (5). For instance, deletion of the Wnt signaling-activated transcription factor Tcf4 (6) or overexpression of the Wnt signaling antagonist Dkk1 (7, 8) resulted in the loss of intestinal crypts, which are the progenitor cell compartments that give rise to the differentiated cells of the villi. Similarly, whereas Wnt3A and Wnt5A can promote self-renewal of hematopoietic stem cells (9–12), the opposite has been observed on addition of the Wnt signaling antagonist Axin (9).

In addition to affecting stem/progenitor cell proliferation, a growing body of evidence suggests that Wnt signaling plays a role in cell fate determination as well as in differentiation. In neural crest stem cells, Wnt1 or chronic activation of the Wnt/ß-catenin pathway has been shown to direct a sensory neuron fate, whereas Wnt3A can promote differentiation into several lineages (13, 14). Likewise, Wnt11 and Wnt5A treatment of hematopoietic progenitor cells favored RBC and monocyte cell fates at the expense of macrophage formation (15). Even more dramatic results have been derived from genetically modified mouse models in which Wnt signaling has been altered in particular tissues, resulting in radical changes in cell fates. For example, constitutive activation of the Wnt pathway in the developing lung in Sftpc-CAtCLef1 transgenic mice impaired the terminal differentiation of the pulmonary epithelium and resulted in the appearance of cells expressing markers of intestinal Paneth and goblet cells (16). Likewise, stabilization of the Wnt signaling effector ß-catenin in the secretory cells of the prostate or mammary gland resulted in squamous metaplasia (17, 18). In skin, interference with Wnt signaling has been shown to adversely affect hair follicle formation by causing cell lineage changes and leads to the development of tumors exhibiting sebaceous differentiation (19, 20). Wnt/ß-catenin signaling is therefore a key mediator of both proliferative and differentiation processes in stem/precursor cell biology, and its misregulation can dramatically alter differentiation and cell fate decisions.

Considering the nature of the aforementioned processes, it is perhaps not surprising that misregulation of Wnt signaling is a hallmark of many forms of cancer, notably including >90% of cases of familial and sporadic colorectal cancers (21). As Wnt signaling pathway components are normally expressed in ovarian granulosa cells (22–24), we have recently investigated the potential involvement of misregulation of this pathway in granulosa cell tumorigenesis (25). We showed that a subset of human and equine granulosa cell tumors (GCT) showed nuclear localization of ß-catenin (the product of the Catnb gene), indicative of inappropriate activation of the Wnt/ß-catenin pathway. To further support the notion that the Wnt/ß-catenin pathway plays a role in the etiology of GCT, mice were genetically engineered to express a dominant stable ß-catenin mutant in granulosa cells. This was accomplished by mating Catnbflox(ex3) mice (which feature a Catnb allele whose third exon is flanked by loxP sites) to the Amhr2cre strain, in which the anti-Mullerian hormone receptor type II coding sequences have been replaced by those encoding Cre recombinase. In the resulting female Catnbflox(ex3)/+;Amhr2cre/+ mice, the Amhr2cre/+ locus drives the expression of Cre in granulosa cells, resulting in the excision of the third exon of Catnb from the floxed allele. The recombined Catnbflox(ex3) allele encodes a ß-catenin protein that, although still functional, lacks a series of phosphorylation sites that are required for its degradation, resulting in its inappropriate accumulation and translocation to the nucleus. Interestingly, Catnbflox(ex3)/+;Amhr2cre/+ mice developed pretumoral lesions by 6 weeks of age, which consisted of follicle-like nests of disorganized, pleiomorphic granulosa cells (termed solid lesions) as well as ovarian cysts. These pretumoral lesions grew no larger than the size of antral follicles and contained very few proliferating cells but often evolved into GCT in older mice. These data showed a causal link between misregulated Wnt/ß-catenin signaling and GCT development and provided a novel model system for the study of GCT biology.

In this study, we have employed the Catnbflox(ex3)/+;Amhr2cre/+ mouse model in an attempt to elucidate the molecular mechanisms by which misregulated Wnt/ß-catenin signaling results in pretumoral lesion and GCT development. Misregulated Wnt/ß-catenin presumably induces tumor formation by altering the expression of specific target genes, the induction and/or repression of which results in abnormal cellular proliferation. We hypothesized that these genes could be identified by profiling gene expression in ovaries from Catnbflox(ex3)/+;Amhr2cre/+ mice bearing pretumoral lesions and comparing these profiles with those of control Catnbflox(ex3)/+ ovaries. Unexpectedly, our results indicated that dominant-stable ß-catenin expression altered the fates of granulosa cells and resulted in multiple metaplasias, suggesting a potentially critical mechanism for GCT development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and serum pleiotrophin measurements. Catnbflox(ex3)/+;Amhr2Cre/+ mice were derived from previously described Amhr2Cre and Catnbflox(ex3) parental strains, and genotyping analyses were done by PCR as described (26, 27). Blood samples were collected by cardiac puncture under anesthesia before euthanasia. Pleiotrophin immunoassays were done on serum as described previously (28). All procedures were approved by the Institutional Animal Care and Use Committee and conformed to the USPHS Policy on Humane Care and Use of Laboratory Animals.

Microarray analysis. Total ovarian RNA was isolated from 12-week-old Catnbflox(ex3)/+;Amhr2Cre/+ and control Catnbflox(ex3)/+ ovaries using the RNeasy Mini kit (Qiagen Sciences, Germantown, MD). At this age, 100% of Catnbflox(ex3)/+;Amhr2Cre/+ ovaries bear pretumoral lesions, but GCT are never observed (25). To avoid ovarian cycle-related differences in gene expression that could arise by comparing single ovaries, RNA samples from six animals per genotype were pooled before microarray probe synthesis. Catnbflox(ex3)/+;Amhr2Cre/+ and Catnbflox(ex3)/+;Amhr2Cre/+ riboprobes were then hybridized to mouse expression set 430 microarrays (Affymetrix, Santa Clara, CA). All steps of RNA quality control, probe synthesis, hybridization, washing, array scanning, and statistical analyses were done by the Microarray Core Facility of the Baylor College of Medicine (Houston, TX).

Semiquantitative reverse transcription-PCR. Reverse transcription-PCR (RT-PCR) was done using the SuperScript One-Step RT-PCR System with Platinum Taq kit (Invitrogen, Carlsbad, CA), and {approx}100 ng samples of ovarian and GCT total RNA that had been isolated as described above. Reactions were formulated as directed by the manufacturer, except that 0.625 µCi [{alpha}-32P]dCTP (specific activity, 3,000 Ci/mmol; MP Biomedicals, Irvine, CA) was added to each reaction to generate quantifiable radioactive signal and to increase assay sensitivity. Oligonucleotides used are detailed in Table 1, except those for ribosomal protein L19 (Rpl19), which were as described previously (ref. 29; Sigma-Genosys, The Woodlands, TX). Cycling conditions were 50°C for 30 minutes and 94°C for 2 minutes followed by a variable number of cycles (as detailed in Table 1; 18 cycles were used for Rpl19) of 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 1 minute. A final extension step of 72°C for 7 minutes was also done. Preliminary experiments were done for all genes assayed to ensure that the cycle numbers selected fell within the linear range of PCR amplification (data not shown). Samples were separated by electrophoresis on 2% TAE-agarose gels, dried, and exposed to Biomax XAR film (Eastman Kodak Co., Rochester, NY) for 1 to 6 hours at –70°C to generate the presented images. The relative radioactive signal strengths from the RT-PCR products were subsequently quantified using a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).


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  		Table 1. Sequences of the oligonucleotide primers employed in the RT-PCR described in the text along with the number of PCR cycles used in the semiquantitative analyses

 
In situ hybridization. In situ hybridization was done on paraformaldehyde-fixed, paraffin-embedded sections of ovaries from 12-week-old Catnbflox(ex3)/+ and Catnbflox(ex3)/+;Amhr2Cre/+ mice as well as GCT isolated from Catnbflox(ex3)/+;Amhr2Cre/+ animals ages 7 to 11 months. Riboprobes were synthesized from cDNA fragments that had been generated by RT-PCR as described above and subsequently cloned into the pCR4-TOPO plasmid vector using the TOPO TA cloning kit (Invitrogen). Probe preparation, slide preparation, hybridization, washing, and exposure/developing steps were done as described previously (30, 31). Exposure times to NTB-2 emulsion (Eastman Kodak) of 2 to 7 days were required to generate the images shown. Control sense riboprobes for each gene failed to generate any specific signal when hybridized under the same conditions (data not shown).

Statistical methods. For the data presented in Fig. 1, unpaired t tests were used to test for differences between groups, with P < 0.05 considered statistically significant. In Fig. 3, one-way ANOVA was employed to test for differences between groups. When differences were identified (P < 0.05), Tukey's test was then used to compare Catnbflox(ex3)/+;Amhr2cre/+ data sets with Catnbflox(ex3)/+ controls at each age examined. Dunnett's test was also employed to test the effects of age on mRNA levels in Catnbflox(ex3)/+;Amhr2cre/+ mice using the 3-week time point data set as a control for comparison with other data sets. One-way ANOVA and Tukey's test were also applied to the pleiotrophin data (Table 2). All tests were done using Prism software version 4.0a (GraphPad Software, Inc., San Diego, CA).


Figure 1
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  		Figure 1. Altered gene expression in the ovaries of Catnbflox(ex3)/+;Amhr2cre/+ mice. Ovarian gene expression analyses comparing Catnbflox(ex3)/+;Amhr2cre/+ ovaries with Catnbflox(ex3)/+ control animals were done using Affymetrix microarrays, and selected genes overexpressed in Catnbflox(ex3)/+;Amhr2cre/+ ovaries (except Rpl19, which was used for standardization) were categorized. Ovarian overexpression of all genes was verified by semiquantitative RT-PCR analyses using six animals per genotype, and results were expressed as the ratios of the means of Rpl19-standardized signal strengths from Catnbflox(ex3)/+;Amhr2cre/+ ovaries to Catnbflox(ex3)/+ controls along with Ps derived from statistical comparison tests between groups. Note that some ratios could not be calculated due to absence of detectable signal in control ovaries. Signal strength ratios and Ps from the original microarray analysis are also shown for comparison. Autoradiographic images illustrate RT-PCR results from three randomly selected animals per genotype.

 

Figure 3
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  		Figure 3. Age-dependent ovarian expression of Wnt signaling inhibitors in Catnbflox(ex3)/+;Amhr2cre/+ and Catnbflox(ex3)/+ mice. Total ovarian RNA was isolated from mice at the indicated ages as well as from GCT obtained from Catnbflox(ex3)/+;Amhr2cre/+ mice. RT-PCR analyses were done to quantify Wnt signaling inhibitor mRNA levels relative to the housekeeping gene Rpl19. Columns, averages of the normalized values; bars, SE. Only single values were obtained at t = 3 days, as RNA samples from five animals per genotype had to be pooled to obtain samples of sufficient size. For other time points, n = 2 (Catnbflox(ex3)/+, t = 3 months), n = 4 (Catnbflox(ex3)/+, t = 3 weeks and 7.5 months; Catnbflox(ex3)/+;Amhr2cre/+, t = 3 months), or n = 3 (all other time points). *, P < 0.05; **, P < 0.01; ***, P < 0.001, statistically significant differences between Catnbflox(ex3)/+;Amhr2cre/+ and Catnbflox(ex3)/+ groups at a given time point. &, P < 0.05, statistically significant time-dependent (or tumorigenesis-dependent) increases in gene expression in Catnbflox(ex3)/+;Amhr2cre/+ animals (using the 3-week time point as a reference). Note that Wif1 mRNA could not be detected at any time point in Catnbflox(ex3)/+ ovaries.

 

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  		Table 2. Serum pleiotrophin measurements, comparing animals of the indicated genotypes and age groups

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ovarian gene expression is altered in Catnbflox(ex3)/+;Amhr2Cre/+ mice. To elucidate putative changes in ovarian gene expression associated with the development of pretumoral lesions in Catnbflox(ex3)/+;Amhr2Cre/+ mice, microarray analyses were done comparing gene expression in Catnbflox(ex3)/+;Amhr2Cre/+ versus Catnbflox(ex3)/+ ovaries from 12-week-old animals. Consistent with the prediction that stabilization of the ß-catenin protein would result in the transcriptional activation of a set of target genes, >40 genes were found to be overexpressed by >2.5-fold in Catnbflox(ex3)/+;Amhr2Cre/+ ovaries (data not shown). Overexpressed genes notably included several known Wnt signaling pathway antagonists, among which Wif1 was the single most highly overexpressed gene identified (Fig. 1A). Unexpectedly, no genes specifically associated with granulosa cell proliferation or biological functions were identified. Rather, most genes that were overexpressed in Catnbflox(ex3)/+;Amhr2Cre/+ ovaries were typically representative of nonovarian tissues and included multiple genes associated with neuronal and neurosecretory-type cells (Fig. 1B) and bone (Fig. 1C). These microarray data were confirmed by semiquantitative PCR, which yielded results comparable with the microarray data (Fig. 1). Microarray analyses also identified a limited number of genes whose expression was modestly decreased in Catnbflox(ex3)/+;Amhr2Cre/+ ovaries relative to Catnbflox(ex3)/+ controls (data not shown), but no genes were found to be underexpressed by more than ~3-fold.

Ectopic gene expression localizes to pretumoral lesions in the ovaries of Catnbflox(ex3)/+;Amhr2Cre/+ mice. In situ hybridization analyses were done to localize the expression of the genes detailed in Fig. 1 in ovaries of normal and Catnbflox(ex3)/+;Amhr2Cre/+ mice. The Wnt signaling antagonists listed in Fig. 1A were undetectable in Catnbflox(ex3)/+ ovaries, with the exception of Axin2 and Nkd1, which localized exclusively to oocytes (Fig. 2A). In Catnbflox(ex3)/+;Amhr2Cre/+ ovaries, all antagonists localized to the pretumoral lesions, with the exception of Dkk4, which was expressed at levels below the detection threshold (Fig. 2A; data not shown). Whereas Axin2 and Nkd1 were detected in all lesions, Wif1 expression was apparently confined to solid pretumoral lesions and could not be detected in cystic-type lesions (Fig. 2A, arrowhead), suggesting a functional difference between the cells that compose cystic and solid lesions. Together, these results indicate that Axin2, Nkd1, and Wif1 are not normally expressed in ovarian granulosa cells and that their expression in Catnbflox(ex3)/+;Amhr2Cre/+ pretumoral granulosa cells likely represents the induction of multiple negative feedback loops to counteract the constitutive activation of the Wnt/ß-catenin pathway.


Figure 2
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  		Figure 2. In situ hybridization analyses of genes of interest in ovaries of 12-week-old Catnbflox(ex3)/+;Amhr2cre/+ and Catnbflox(ex3)/+ mice localize their expression to pretumoral lesions. Sections in bright-field views are counterstained with hematoxylin; radioactive signal appears as light-colored grains in dark-field views. Short arrows, oocytes; long arrows, examples of solid pretumoral lesions; arrowheads, cystic lesions. Original magnifications, x50. A, analysis of Wnt signaling inhibitor expression. Ovarian sections from animals of the indicated genotypes were hybridized with the indicated riboprobes. Note that the dark-field view for Axin2 in the Catnbflox(ex3)/+ section was overexposed to enhance the weak, oocyte-specific signal. No signal was detected for Wif1 in Catnbflox(ex3)/+ ovaries. B, analysis of Cdkn1c and Ptn expression in Catnbflox(ex3)/+;Amhr2cre/+ ovaries. No signal was detected for either gene in Catnbflox(ex3)/+ controls (data not shown).

 
Of the neuronal/neurosecretory and bone genes listed in Fig. 1B and C, only Ptn and Cdkn1c were detectable by in situ hybridization in Catnbflox(ex3)/+;Amhr2Cre/+ ovaries, and none were detectable in Catnbflox(ex3)/+ controls (Fig. 2B; data not shown). As for Axin2 and Nkd1, Ptn was found to be expressed in all Catnbflox(ex3)/+;Amhr2Cre/+ pretumoral lesions. Although Cdkn1c was also found in pretumoral lesions, it showed a more mottled, patchy expression pattern and great variability in expression levels between lesions, suggesting that only a small subset of cells within the lesions expressed Cdkn1c (Fig. 2B).

Time-dependent effects of pretumoral lesion and GCT formation on Wnt antagonist expression in Catnbflox(ex3)/+;Amhr2Cre/+ ovaries. To study the temporal changes in expression of the Wnt signaling antagonists Axin2, Nkd1, and Wif1 in ovaries of Catnbflox(ex3)/+;Amhr2Cre/+ mice, ovaries were harvested from 3-day and 3-week-old mice (i.e., before pretumoral lesion development), from 6-week-old, 3-month-old, and 7.5-month-old mice (i.e., with pretumoral lesions but before GCT development), and from mice with GCT. Semiquantitative RT-PCR analyses showed similar patterns of expression for Axin2 and Nkd1. They were found to be expressed at high and comparable levels in both Catnbflox(ex3)/+;Amhr2Cre/+ and control Catnbflox(ex3)/+ ovaries in 3-day-old mice (Fig. 3). As both genes are expressed in oocytes (Fig. 2A), this was likely reflective of the oocyte-rich composition of the developing ovary in neonatal mice. In Catnbflox(ex3)/+ controls, ovarian Axin2 and Nkd1 mRNA levels then dropped by 3 weeks as ovarian development progressed and the oocyte/ovarian stroma ratio decreased and then remained relatively constant throughout adulthood. However, in Catnbflox(ex3)/+;Amhr2Cre/+ ovaries, Axin2 and Nkd1 transcripts were found to be significantly more abundant ({approx}4- to 12-fold) than in age-matched controls as early as 3 weeks of age and continuing through adulthood. Axin2 and Nkd1 expression increased further in GCT, and mRNA levels were found to be significantly (albeit modestly, {approx}2-fold) higher than at 3 weeks, which was more likely reflective of the loss of normal ovarian tissue than a tumorigenesis-associated increase in expression in the granulosa cells. A different pattern of expression was observed for Wif1, as its expression was not detected at any time in Catnbflox(ex3)/+ controls (Fig. 3). It was, however, detectable in Catnbflox(ex3)/+;Amhr2Cre/+ ovaries at 3 days and its expression increased rapidly by 3 weeks and attained highest levels in the oldest mice with pretumoral lesions and mice with GCT. This pattern of expression closely mirrored that of {Delta}ex3 Catnb mRNA (i.e., the transcript derived from the Cre-recombined Catnb allele) in Catnbflox(ex3)/+;Amhr2Cre/+ ovaries (25). Together, these data indicate that Wnt signaling antagonist expression in Catnbflox(ex3)/+;Amhr2Cre/+ ovaries is initiated in parallel with Cre-mediated recombination well before the appearance of the pretumoral lesions and that pretumoral lesion and GCT formation occurs despite the uninterrupted presence of their Wnt signaling antagonist activities. This suggests that these genes are incapable of completely repressing misregulated Wnt/ß-catenin signaling in Catnbflox(ex3)/+;Amhr2Cre/+ ovaries and that their silencing is not required for later development of GCT.

Expression of bone and neurosecretory cell markers in distinct cell populations in Catnbflox(ex3)/+;Amhr2Cre/+ GCT reveals the presence of distinct metaplastic cell types. To study the expression of the genes described in Fig. 1 in GCT from Catnbflox(ex3)/+;Amhr2Cre/+ ovaries, in situ hybridization analyses were done for each gene using a panel of tumors. Results showed the presence of nests of neoplastic granulosa cells within many GCT that expressed all Wnt signaling antagonists detected in the pretumoral lesions (Fig. 4A). These cells also expressed the neuronal marker Ptn as well as Cck, which encodes the neuropeptide cholecystokinin. A distinct, less common cell population was found to express the bone marker Ibsp (Fig. 4B). Areas of Ibsp expression failed to show expression of the Wnt antagonist or neurosecretory cell markers described in Fig. 4A and were invariably associated with foci of ossification. Together with the data showing bone marker expression in pretumoral lesions before GCT development and before obvious bone formation (Figs. 1C and 2B), these results show that stabilization of ß-catenin induces osseous metaplasia in ovarian granulosa cells. Likewise, the presence of Cck-positive populations in the GCT, in addition to the expression of neuronal/neurosecretory markers in the pretumoral stages (Figs. 1B and 2A), is suggestive of neuronal metaplasia. Although the presence of cholecystokinin-positive nerve fibers has been reported in the interstitium of the normal ovary (32, 33), these are thought to originate from neurons situated in the dorsal root ganglia rather than an endogenous ovarian neurosecretory cell population, which is supported by our inability to detect Cck mRNA in normal ovaries (Fig. 1B). It is therefore unlikely that the tumoral Cck-positive cells represent a neoplastic proliferation of ovarian neurosecretory cells. Indeed, the tumoral Cck-positive cells morphologically resemble granulosa cells, supporting the concept that they are of granulosa cell origin and are undergoing metaplastic transformation.


Figure 4
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  		Figure 4. In situ hybridization analyses of genes of interest in GCT from Catnbflox(ex3)/+;Amhr2cre/+ mice reveal the presence of distinct metaplastic cell types. A to C, results from the hybridization of the riboprobes described in Table 1 to three different tumors, respectively. Although the same field is shown in all images of each panel, slight differences in tissue morphology occur from one image to the next due to the use of nonconsecutive histologic sections. Original magnifications, x50, except as indicated (H&E stain). A, dark-field views of sections hybridized to the indicated riboprobes. In all cases, signals corresponded to nests of tumorous granulosa cells as shown in the bright-field image (top left). GC, granulosa cells. B, a region of metaplastic ossification ({circ}) hybridized to the Ibsp riboprobe but not to the markers characteristic the cell type defined in (A), which are also seen surrounding the ossified region. The bright-field view of the hematoxylin-stained section hybridized to Ibsp is shown to illustrate tissue morphology. C, a third, spindle-shaped cell type that was typically found at the boundaries of ossified regions ({circ}) is shown at low and high magnification (arrows). These cells hybridized specifically to the Cdkn1c riboprobe but not to the other markers shown in (A and B).

 
A third, spindle-shaped cell population was typically found immediately adjacent to foci of ossification (Fig. 4C, arrows). Unlike the other cell types, these were found to be strongly positive for Cdkn1c (which encodes the cell cycle inhibitor p57Kip2) and no other markers were examined. As Cdkn1c expression is induced in osteoblast precursors, as these cease proliferating and undergo differentiation (34), the Cdkn1c-positive cells may represent a differentiating precursor subpopulation of the metaplastic bone cells. This is supported by the presence of Cdkn1c-positive cells in pretumoral lesions (Fig. 2B) before bone formation and before Ibsp can be detected by in situ hybridization. Although circumstantial, the localization of the Cdkn1c-positive population between the Cck-positive cell population and the foci of osseous metaplasia could indicate that the former may give rise to the latter or vice versa.

Serum pleiotrophin levels in Catnbflox(ex3)/+;Amhr2cre/+ mice. The cytokine pleiotrophin, the product of the Ptn gene, has recently been found at increased levels in serum from patients with several tumor types, suggesting its potential use as a diagnostic or prognostic marker (refs. 35–37). Our observation that Ptn was highly expressed in both the pretumoral lesions and GCT in Catnbflox(ex3)/+;Amhr2cre/+ mice (Figs. 1B, 2B, and 4) raised the possibility that serum pleiotrophin levels could also be increased in this model and by extension perhaps in human GCT. To test this, serum pleiotrophin levels were measured in both young (6-12 weeks) Catnbflox(ex3)/+;Amhr2cre/+ mice bearing pretumoral lesions and older (>24 weeks) mice susceptible to developing GCT as well as in age-matched Catnbflox(ex3)/+ controls. The highest levels of pleiotrophin were identified in two >24-week-old Catnbflox(ex3)/+;Amhr2cre/+ mice bearing large GCT (12.42 and 22.88 ng/mL, respectively). However, serum pleiotrophin levels comparable with controls were detected in the vast majority of Catnbflox(ex3)/+;Amhr2cre/+ mice, and no statistically significant differences could be detected between experimental groups (Table 2).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Metaplasia, defined simply as the transformation of one tissue type to another, is a poorly understood process that has been observed in a wide range of tumor types (38–40). Although the relevance of metaplasia to the disease process remains unclear in most tumors, in some cases, it is thought to represent an essential premalignant step in the series of events that give rise to cancer. Perhaps the best-known example of this is Barrett's esophagus, a condition in which the squamous epithelium of the lower esophagus is replaced by intestinal epithelium, which predisposes the patient to develop esophageal adenocarcinoma (40). In this study, we report that changes in gene expression indicative of both osseous and neuronal metaplasia occur in the ovarian pretumoral lesions in the Catnbflox(ex3)/+;Amhr2cre/+ model and that the GCT that develop later in life consist (at least in part) of clonal expansions of these metaplastic cell types. To our knowledge, this is therefore the first report to show a metaplastic intermediate step in the etiology of GCT. Furthermore, although osseous metaplasia has long been known to occur in a variety of tumor types (38, 39), the molecular mechanisms underlying its development are completely unknown. Our results indicate that constitutive activation of the Wnt/ß-catenin pathway results in metaplastic ossification in the ovary, and the Catnbflox(ex3)/+;Amhr2cre/+ mouse could serve as a model for the study of this poorly understood phenomenon. Whether Wnt/ß-catenin signaling defects are involved in the etiology of metaplastic ossification in other tissue and tumor types is an important question that remains to be resolved.

The exact origin of the metaplastic cells that form the pretumoral lesions in Catnbflox(ex3)/+;Amhr2cre/+ ovaries remains unclear. Some extent of Cre-mediated genetic recombination is thought to occur prenatally/perinatally in the ovaries of Catnbflox(ex3)/+;Amhr2cre/+ mice (25), which is supported by our observation of ectopic Wif1 expression in the ovaries of animals as young as 3 days. Recombination leads to the appearance of visibly discernable pretumoral lesions by the 5th or 6th week of life, and these do not seem to grow in number thereafter despite the presence of abundant normal-looking follicles whose granulosa cells predictably express Cre (25, 27). The prenatal/perinatal phase therefore seems to represent the critical time period for the formation of the pretumoral cells, after which these proliferate to form pretumoral lesions, but few, if any, additional "founder" pretumoral cells are created. The earliest events of follicular development also occur during this time in the normal mouse ovary, as ovarian somatic cells proliferate, differentiate into granulosa cell precursors, and become organized with oocytes to form primordial follicles (41). Given this and the known effect of misregulated Wnt signaling on cell fate decisions in other cell types (16–20), it would seem reasonable to propose that the pretumoral cells could arise from a multipotent ovarian somatic stem/progenitor cell type that undergoes genetic recombination early during ovarian development (Fig. 5A). Metaplasia then results, as misregulated Wnt/ß-catenin signaling causes these progenitor cells to commit to inappropriate lineages, a process that Okubo and Hogan have recently called transdetermination (16). Alternatively, cells that are committed to become granulosa cells may have their fates altered on genetic recombination and Wnt/ß-catenin signaling activation, a mechanism that many authors now call transdifferentiation (16, 42). Although our experimental design could not permit us to distinguish between these two scenarios, we believe transdifferentiation to be less likely than transdetermination, as the granulosa cells present in normal-looking follicles found in adult Catnbflox(ex3)/+;Amhr2cre/+ ovaries seem incapable of undergoing metaplastic changes following commitment to the granulosa cell fate.


Figure 5
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  		Figure 5. Cellular and molecular processes in Catnbflox(ex3)/+;Amhr2cre/+ granulosa cells. A, theoretical model illustrating the proliferation and differentiation dynamics of ovarian granulosa cells and their precursors in normal ovaries versus Catnbflox(ex3)/+;Amhr2cre/+ pretumoral and tumoral lesions. Metaplastic cell types may arise either by transdetermination (i.e., commitment of a multipotent stem/precursor cell type to an inappropriate lineage) or by transdifferentiation (i.e., alteration of the fate of a cell already committed to a particular lineage). B, misregulated Wnt/ß-catenin signaling induces multiple, complementary negative feedback loops. The level at which each signaling antagonist described in Fig. 1 affects the canonical Wnt signaling pathway is illustrated. Axin2 transcription is activated by TCF/LEF family transcription factors (44) and is a direct target of the canonical Wnt/ß-catenin signaling pathway; whether this is true for the other antagonists has not been reported.

 
Our results indicate that Axin2 and Nkd1 are expressed in oocytes, and the expression of these two genes was also observed in pretumoral lesions and the GCT Cck-positive cell population in Catnbflox(ex3)/+;Amhr2cre/+ mice. Considering the pluripotency of tumoral germ cells (as observed in teratomas in particular) and the pluripotent characteristics of the GCT in the Catnbflox(ex3)/+;Amhr2cre/+ model, this would suggest that one or more cell types observed in Catnbflox(ex3)/+;Amhr2cre/+ GCT may be oocyte derived. However, the Cck-positive cell population morphologically resembles granulosa cells, and extensive histopathologic analyses showed that all ovarian tumors observed in Catnbflox(ex3)/+;Amhr2cre/+ mice to be GCT, without bearing any resemblance to teratomas or any other type of germ cell tumor (25). Furthermore, direct targeting of ß-catenin stabilization to oocytes using Catnbflox(ex3) and Tg(Zp3-cre) mice failed to generate germ cell tumors.4 Therefore, although an oocyte contribution to the GCT observed in Catnbflox(ex3)/+;Amhr2cre/+ mice cannot be ruled out, we believe that the vast majority of the cells in these tumors arise from a somatic stem/progenitor cell type as stated above.

Another important finding reported herein is the induction of the expression of multiple antagonists of Wnt/ß-catenin signaling in Catnbflox(ex3)/+;Amhr2cre/+ ovarian pretumoral and tumoral cells. Axin2, Dkk4, Nkd1, and Wif1 each interact with a different component of the Wnt/ß-catenin signaling cascade, indicating the existence of multiple, nonredundant feedback loops that could act in a coordinate manner to shutdown the Wnt/ß-catenin pathway at many levels simultaneously (Fig. 5B). Although the involvement of each of these Wnt antagonists in negative feedback loops has been suggested in previous reports (43–48), the concept that multiple loops could act coordinately within a given cell is unusual. As the activation of the Wnt/ß-catenin pathway in Catnbflox(ex3)/+;Amhr2Cre/+ mice occurs at the level of ß-catenin itself and all of the negative feedback loops act upon or upstream of ß-catenin, all of these mechanisms predictably fail to inhibit pathway activity in this model. Indeed, as Wnts can signal via at least two additional pathways (the Wnt/Ca2+ and planar cell polarity pathways; ref. 2), many of the negative feedback loops could conceivably be harmful, contributing to the pathogenesis by shutting down potentially beneficial Wnt signals that are transduced via ß-catenin-independent mechanisms.

Of the genes that are induced in Catnbflox(ex3)/+;Amhr2cre/+ ovaries, Ptn may be of particular pathophysiologic relevance to both metaplasia and tumorigenesis phenotypes. We have listed Ptn as a neuronal marker as it is expressed in specific areas of the developing and adult nervous system (49), Ptn-deficient mice exhibit neurophysiologic and behavioral phenotypes (50, 51), and it is thought to affect the proliferation and differentiation of particular neuronal cell types (52, 53). However, Ptn has also been proposed to play important roles in bone physiology, specifically as a positive regulator of both osteoblast differentiation and function (54, 55). The induction of Ptn could thus conceivably influence the lineages to which pretumoral cells are diverted in Catnbflox(ex3)/+;Amhr2cre/+ ovaries, leading to the observed osseous and neuronal metaplasias. Furthermore, pleiotrophin is thought to be a positive regulator of both physiologic and tumoral angiogenesis (56, 57). This may relate to the highly vascularized organization of the pretumoral lesions in Catnbflox(ex3)/+;Amhr2cre/+ ovaries and of GCT in all species (25, 58), which contrasts sharply with the normally avascular granulosa cell layer of ovarian follicles. Beyond angiogenesis, Ptn is believed to contribute to carcinogenesis in a variety of additional ways in many types of cancer, affecting processes as diverse as apoptosis, mitosis, transformation, and chemotaxis (57). Expression of Ptn in certain tumors correlates with increased levels of circulating pleiotrophin, leading several authors to suggest that serum pleiotrophin measurements may be of diagnostic or prognostic value in a clinical setting (35–37). Our serum pleiotrophin results in the Catnbflox(ex3)/+;Amhr2cre/+ mouse show that increased circulating pleiotrophin levels are occasionally found with advanced age following GCT development. However, the infrequency of this observation suggests that pleiotrophin measurement may not be a useful tool for GCT diagnosis. Whether this will be the case for human GCT patients will be grounds for further research.

In summary, we have used microarray analyses to elucidate the molecular processes underlying pretumoral lesion and GCT development in Catnbflox(ex3)/+;Amhr2cre/+ mice. Our results show that constitutive activation of the Wnt/ß-catenin pathway during ovarian development leads to the appearance of distinct metaplastic cell types, suggesting that metaplasia is a key intermediate step in the development of GCT in this model. This study further illustrates the effect of the Wnt/ß-catenin pathway on cell fate decision-making processes and provides insight into how its misregulation can lead to tumor development.


   Acknowledgments
 
Grant support: NIH grants HD16272 and HD07495 (J.S. Richards), Association pour la Recherche sur le Cancer, France grant 3242 (J. Courty), and Canadian Institutes of Health Research fellowship (D. Boerboom).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Yuet K. Lo and Laura Liles for important technical contributions to the work and Drs. Marilene Paquet and Hubert Burden for helpful discussions.


   Footnotes
 
4 D. Boerboom and J.S. Richards, unpublished observations. Back

Received 9/28/05. Revised 11/ 5/05. Accepted 12/13/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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