Cell Type–Specific Targeted Mutations of Kras and Pten Document Proliferation Arrest in Granulosa Cells Versus Oncogenic Insult to Ovarian Surface Epithelial Cells

Endocrinology

Cell Type–Specific Targeted Mutations of Kras and Pten Document Proliferation Arrest in Granulosa Cells versus Oncogenic Insult to Ovarian Surface Epithelial Cells

Heng-Yu Fan,1 Zhilin Liu,1 Marilene Paquet,2 Jinrong Wang,1 John P. Lydon,1 Francesco J. DeMayo,1 and JoAnne S. Richards1

1Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas and 2Comparative Medicine and Animal Resources Center, McGill University, Montreal, Quebec, Canada

Abstract prevalent, accounting for f90% of all human epithelial ovarian The small G-protein KRAS is crucial for mediating gonado- cancers and have been classified based on histologic criteria tropin-induced events associated with ovulation. However, as serous, endometrioid, clear cell, and mucinous. By contrast, constitutive expression of KrasG12D in granulosa cells dis- only 5% of human ovarian cancers are derived from granulosa cells rupted normal follicle development leading to the persistence (1–3). This seems unexpected because granulosa cells are the most of abnormal follicle-like structures containing nonmitotic abundant proliferative cell type in the ovary. It is not known if the cells. To determine what factors mediate this potent effect of low incidence of granulosa cell tumors (GCT) is due to the strong KrasG12D, gene profiling analyses were done. We also analyzed resistance of granulosa cells to oncogenic stimuli, the rare KrasG12D;Cyp19-Cre and KrasG12;Pgr-Cre mutant mouse models occurrence of oncogenic mutations in this population of ovarian that express Cre prior to or after the initiation of granulosa cells, or to the natural fate of granulosa cells that are removed from G12D the proliferative stage either by apoptosis or the natural cell differentiation, respectively. Kras induced cell cycle arrest in granulosa cells of the KrasG12D;Cyp19-Cre mice but progression to terminally differentiated nondividing luteal cells. not in the KrasG12D;Pgr-Cre mice, documenting the cell The proliferation and differentiation of granulosa cells in context–specific effect of KrasG12D. Expression of KrasG12D developing follicles are regulated by the pituitary gonadotropins silenced the Kras gene, reduced cell cycle activator genes, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and impaired the expression of granulosa cell and oocyte- and their G protein–coupled receptors as well as by ovarian-derived specific genes. Conversely, levels of PTEN and phosphorylated growth-regulatory factors (4, 5). Recent studies document that in p38 mitogen-activated protein kinase (MAPK) increased addition to the canonical cyclic AMP/protein kinase A pathway, markedly in the mutant granulosa cells. Because disrupting FSH and LH activate several other signaling cascades such as the Pten in granulosa cells leads to increased proliferation and RAF1/MEK/MAPK3/1 pathway (also known as ERK1/2) and the survival, Pten was disrupted in the KrasG12D mutant mice. phosphoinositide-3-kinase (PI3K)/AKT pathway (6–9) via activation of the small G-protein RAS and the tyrosine kinase SRC, respectively The Pten/Kras mutant mice were infertile but lacked granulosa G12D cell tumors. By contrast, the Ptenfl/fl;KrasG12D;Amhr2-Cre mice (7). Conditional expression of an oncogenic Kras mutation in granulosa cells affect both the ERK1/2 and PI3K pathways and leads developed aggressive ovarian surface epithelial cell tumors fl/fl G12D fl/fl to defects in follicle development and ovulation (10). Surprisingly, that did not occur in the Pten ;Kras ;Cyp19-Cre or Pten ; G12D KrasG12D;Pgr-Cre mouse strains. These data document unequiv- whereas Kras mutations induce tumors in the lung (11), mammary glands (12), and uterus,3 it does not stimulate the ocally that Amhr2-Cre is expressed in and mediates allelic G12D recombination of oncogenic genes in ovarian surface epithelial oncogenic transformation of granulosa cells. Rather, the Kras cells. That KrasG12D/Pten mutant granulosa cells do not expressing granulosa cells undergo cell cycle arrest, are nonmitotic transform but rather undergo cell cycle arrest indicates that based on the absence of either bromodeoxyuridine (BrdU) labeling they resist the oncogenic insults of Kras/Pten by robust or phosphorylated histone H3 immunostaining, are nonapoptotic self-protecting mechanisms that silence the Kras gene and based on the lack of terminal nucleotidyl transferase–mediated nick elevate PTEN and phosphorylated p38 MAPK. [Cancer Res end labeling–positive cells or cells exhibiting cleaved caspase 3 2009;69(16):6463–72] immunostaining, and fail to differentiate (10). The abnormal follicle-like structures are devoid of oocytes and persist in the mutant ovaries for prolonged periods of time (10). Introduction The PI3K pathway is well-recognized to be essential for cell Ovarian cancer represents a major threat to women’s health survival and proliferation; its hyperactivation is associated with because our knowledge of the underlying causes of this complex oncogenesis in multiple tissue and cell types (13, 14). The and heterogeneous disease remains ill-defined. Carcinomas of the phosphatidylinositol phosphate phosphatase PTEN (phosphatase ovarian surface epithelium (OSE) are the most aggressive and tensin homologue) negatively regulates the activity of PI3K pathway and therefore functions as a potent tumor repressor. Although mutations of the Pten locus can lead to tumor formation in many but Note: Supplementary data for this article are available at Cancer Research Online not all tissues (15, 16), the disruption of the Pten gene causes (http://cancerres.aacrjournals.org/). Requests for reprints: JoAnne S. Richards, Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030. Phone: 713-798-6259; Fax: 713-790-1275; E-mail: [email protected]. I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-3363 3 J. Wang and F. DeMayo, unpublished results. www.aacrjournals.org 6463 Cancer Res 2009; 69: (16). August 15, 2009

Figure 1. KrasG12D selectively affects granulosa cell function in mice expressing Cre driven by the Amhr2 (A-Cre) and Cyp19 (C-Cre) but not Pgr (P-Cre) promoters. Ovaries of KrasG12D;A-Cre mice accumulate more small abnormal follicle-like lesions (arrows) from 1 to 6 mo of age than did the KrasG12D;C-Cre mice. No abnormal follicles were observed in ovaries of the KrasG12D;Pgr-Cre mice. NF, normal follicles; CL, corpora luteum.

embryonic lethality in mice, indicating that it is also essential for K-rasG12D parental strains, and genotyping was done by PCR as described normal development (17). Selective depletion of Pten in mouse (10, 20–23). granulosa cells leads to hyperactivation of the PI3K pathway, Histologic images and the efficient recombination of the mutant alleles increased follicle growth and ovulation, and the prolonged life span was documented for each genotype (Supplementary Fig. S2G). Animals were housed under a 16-h light/8-h dark schedule in the Center for Comparative of corpora lutea (18) but rarely induces GCTs (19). Medicine at Baylor College of Medicine and provided food and water In this study, we sought to determine the cell-specific and G12D ad libitum. Animals were treated in accordance with the NIH Guide for the combinatorial effects of expressing Kras and mutant Pten in Care and Use of Laboratory Animals, as approved by the Animal Care and mouse ovarian cells. Using six different mouse models, we document Use Committee at Baylor College of Medicine. G12D that the Kras mutation in granulosa cells induces cell cycle Immature 21-day-old female mice were injected i.p. with 4 IUof arrest in a developmental stage–dependent manner and that mice equine chorionic gonadotropin (Calbiochem) to stimulate follicular with double mutations of Pten/Kras selectively in granulosa cells growth, followed 48 h later with 5 IUof human chorionic gonadotropin were infertile but showed no GCTs. By contrast, in the Ptenfl/fl; (hCG; American Pharmaceutical Partners) to stimulate ovulation and KrasG12D;Amhr2Cre mice, in which double mutations are expressed luteinization. Ovulated oocytes were collected from oviducts 16 h after in granulosa cells and the OSE cells, massive ovarian serous papillary hCG injection. The development of ovarian tumors was checked in mice at the ages of 1, 3, and 6 mo. adenocarcinomas develop at an early age. These results indicate that Histology and immunohistochemistry. Ovaries were collected and granulosa cells are highly resistant to these oncogenic factors that fixed in 4% paraformaldehyde and embedded in paraffin and processed by transform OSE cells and therefore possess robust self-protecting routine procedures (10). Immunohistochemistry was done as previously mechanisms that arrest cell cycle progression. described (10) using antibodies against cytokeratin 8 (Abcam), PTEN (Cell Signaling), or proliferating cell nuclear antigen (Cell Signaling). Sections were counterstained with hematoxylin, dehydrated, and mounted. Materials and Methods Immunofluorescence. Ovaries were fixed in 4% paraformaldehyde, Animals. LSL-KrasG12D;Amhr2-Cre, LSL-KrasG12D;Cyp19-Cre, LSL- embedded in optimal cutting temperature compound (Sakura Finetek USA, KrasG12D;Pgr-Cre, Ptenfl/fl;LSL-KrasG12D;Amhr2-Cre, Ptenfl/fl;LSL-KrasG12D; Inc.) and stored at À70jC before the preparation of 7 Amol/L sections and Cyp19-Cre,andPtenfl/fl;LSL-KrasG12D;Pgr-Cre mice were derived from immunostaining (10). Antibodies for WT1, KRAS, cyclin A, and E2F1 were previously described Amhr2-Cre, Cyp19-Cre, Pgr-Cre, Ptenfl/fl and LSL- purchased from Santa Cruz Biotechnology. All the other antibodies were

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Cell-Selective Effects of Kras and Pten in the Ovary purchased from Cell Signaling. The MUC16 antibody was a gift from RNeasy Mini kit (Qiagen Sciences). At 3 mo of age, 100% of the Dr. Robert Bast, Jr., (Department of Experimental Therapeutics, M.D. KrasG12D;Amhr2-Cre ovaries contained an abundance of abnormal follicles, Anderson Cancer Center, University of Houston, Houston, TX). but not corpora lutea, whereas the immature mouse ovaries contained BrdU incorporation assay. BrdUrd and a monoclonal antibody against predominantly normal follicles. Thus, this was deemed the most BrdU(Sigma Chemical Company) were used as previously described (10). appropriate comparison for detecting granulosa cell genes. To avoid Hormone assays. RIAs for FSH and estradiol were performed by ovarian-related differences in gene expression that could arise by the Ligand Assay and Analysis Core, Specialized Cooperative Centers comparing single ovaries, RNA samples from three animals per genotype Program in Reproduction and Infertility Research (University of Virginia), were pooled prior to microarray probe synthesis. Wild-type and KrasG12D; as previously described (24). Amhr2-Cre riboprobes were then hybridized to Mouse 430.2 microarray Microarray analyses. Total ovarian RNA was isolated from 3-mo-old chips (Affymetrix). All steps for RNA quality control, probe synthesis, KrasG12D;Amhr2-Cre mice and immature 26-d-old control mice using the hybridization, washing, array scanning, and statistical analyses were done

Figure 2. KrasG12D down-regulates KRAS and up-regulates PTEN in the follicle-like lesions. Granulosa cells of normal growing follicles express KRAS whereas KRAS was negligible in cells within the abnormal follicles (A; arrows). NF, normal follicles. PTEN levels were low in granulosa cells of normal follicles but were elevated in the mutant granulosa cells (B; arrows) and this was associated with reduced levels of phosphorylated AKT (C; wild-type versus KrasG12D;A-Cre) but high levels of phosphorylated p38 MAPK (D; pMAPK).

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Cancer Research by the Microarray Core Facility of the Baylor College of Medicine (Houston, documented that genes regulating cell cycle progression (27), TX) as previously described (25). especially cyclins and members of the E2F transcription factor RNA isolation and real-time PCR. For real-time reverse transcription- family, were down-regulated, whereas the genes, Pten and Zbtb16 PCR analyses, additional total ovarian RNA was also isolated from ovaries of (also known as Plzf ), which have been reported to repress cell cycle control (26 d old and 3 mo old) mice, KrasG12D;Amhr2-Cre (3 mo old) mice, progression (28), were up-regulated (Supplementary Table S1; and Ptenfl/fl;KrasG12D;Amhr2-Cre (3 mo old) mice. Reverse transcription was done using the SuperScript One-step RT-PCR system with Platinum Taq kit Fig. 3B). Genes that control granulosa cell differentiation [FSH (Invitrogen). The real-time PCR was performed using the Rotor-Gene 3000 receptor (Fshr), activin/inhibin (Inhba/Inhbb/Inha), and aromatase thermocycler (Corbett Research, Sydney, Australia). The PCR reaction (Cyp19)] and genes encoding key transcription factors Fos, Foxo1, G12D included 5 AL of SYBR Green JumpStart Taq Mix (Sigma), 4.0 AL of first- and Nr5a2 were down-regulated in the Kras mutant cells strand cDNA product described above (1:100 dilution), and 0.5 Amol/L of (Supplementary Table S1). By contrast, expression levels of the forward and reverse primers. Relative levels of gene expression were LH receptor (Lhcgr) and the cholesterol side chain cleavage calculated using Rotor-Gene 6.0 software and normalized to h-actin. cytochrome P450 enzyme (Cyp11a1) increased, and are likely Western blot analysis. Tissues and cells were lysed with radio- associated with the hypertrophy of interstitial cells in the Kras immunoprecipitation assay buffer containing complete protease inhibitors mutant ovaries. (Roche; ref. 10). Western blots were performed using 30 Ag of lysate protein, KrasG12D-mediated cell cycle arrest in ovarian granulosa incubated with antibodies specific for FOXO1, FOXO3, PTEN, AKT, cyclin D2, and phosphorylated mTOR, GSK3h, PDK1, ERK1/2, and AKT (1:1,000 cells is associated with elevated PTEN and phosphorylated p38 dilution; all from Cell Signaling Technology), cyclin A (Santa Cruz mitogen-activated protein kinase and reduced expression of Biotechnology), and E2F1 (Santa Cruz Biotechnology). KRAS. KRAS is highly expressed in granulosa cells of normal Statistical analyses. The data for real-time reverse transcription-PCR growing follicles (Fig. 2A, WT), but was undetectable in the assays, breeding experiments, hormone levels, and super-ovulation tests are nonmitotic granulosa cells within the abnormal follicles (Fig. 2A, represented as mean F SD. Data were analyzed by using GraphPad Prism Kras;Amhr2-Cre). Moreover, the response of granulosa cells to Programs (ANOVA or t test; GraphPad Prism) to determine significance. KRASG12D is dependent on the mutation level of the KRAS V V Values were considered significantly different if P 0.05 or P 0.01. expressed in these cells (Supplementary Fig. S1A–C), supporting the evidence that p15INK4B, p16INK4A, and TRP53 are unlikely to mediate the effects of KRASG12D in granulosa cells of the Results Kras;Amhr2-Cre mice in vivo. KrasG12D induction of cell cycle arrest in ovarian granulosa Because the microarray results indicated that Pten mRNA levels cells is stage-dependent. The KrasG12D;Amhr2-Cre mutant mice increased in the Kras mutant ovaries, the cell-specific expression of contain abnormal follicle-like structures consisting of nonmitotic PTEN protein was analyzed and shown to be low in granulosa cells and nonapoptotic granulosa cells that persist for prolonged periods of the growing follicles present in ovaries of control mice compared of time (10). As shown herein, the ovaries of KrasG12D;Amhr2-Cre with interstitial cells and corpora lutea (Fig. 2B, WT; ref. 18). In mice progressively increase in size as the mice age and this is marked contrast, PTEN was dramatically elevated in cells within associated with the accumulation of the small follicle-derived the follicle lesions present in ovaries of the Kras mutant mice ovarian lesions (Fig. 1A, for each time point or genotype, ovaries (Fig. 2B, Kras;Amhr2-Cre; Supplementary Fig. S4). Elevated PTEN in from at least four animals were sectioned, and ovarian lesions the mutant granulosa cells were associated with negligible immu- were counted in six sections of each ovary). To determine if the nostaining for phosphorylated AKT compared with granulosa cells in formation of these abnormal follicle-like structures was dependent normal growing follicles (Fig. 2C). In contrast to the suppression of the on the stage of granulosa cell differentiation, we generated three KRAS-ERK1/2 and AKT pathways in the KrasG12D mutant granulosa KrasG12D mutant mouse models using mouse strains (Amhr2-Cre, cells, elevated levels of phosphorylated p38 mitogen-activated protein Cyp19-Cre and Pgr-Cre) in which Cre is driven by promoters kinase (pMAPK14) were observed in these cells (Fig. 2C). Although expressed at different stages of follicular development in vivo.In phosphorylated p38 MAPK is commonly associated with oncogenic- the Amhr2-Cre and Cyp19-Cre females, Cre-mediated allelic induced cell cycle arrest and senescence in fibroblasts and epithelial recombination occurs primarily in proliferating granulosa cells of cells (29, 30), the KrasG12D mutant granulosa cells did not express primary follicles and early antral follicles, respectively. Because the senescence-associated (SA)-h-galactosidase (31) or the secretory expression of the endogenous progesterone receptor (Pgr) gene is factor, interleukin-6 (ref. 32; data not shown). Thus, the complete induced only in granulosa cells of preovulatory follicles exposed to silencing of the Kras gene (Fig. 2A) and down-regulation of the cell the LH surge, Cre-mediated allelic recombination in the Pgr-Cre cycle regulatory molecules (Supplementary Table S1) combined with mice is restricted to terminally differentiating granulosa cells up-regulation of PTEN and phosphorylated p38 MAPK seem to undergoing luteinization (Fig. 1B; refs. 23, 26). When the ovaries of characterize the phenotype in the cycle arrested granulosa cells. the three mutant mouse models were compared at 6 months of Double mutations of Pten and Kras in granulosa cells do not age, the ovaries of KrasG12D;Amhr2-Cre females contained 2-fold lead to tumors but do disrupt ovarian function and fertility more abnormal follicle-like structures than those of the KrasG12D; leading to premature ovarian failure. Because disrupting Pten Cyp19-Cre mice (Fig. 1A). Strikingly, ovaries of the KrasG12D;Pgr-Cre alone in ovarian granulosa cells in the Ptenfl/fl;Cyp19-Cre mice females contained no abnormal follicle-like structures and were increases fertility and leads to the extended survival and life span of normal (Fig. 1B), indicating that the KrasG12D mutation initiates granulosa/luteal cells (18) and only rarely leads to GCTs in the inappropriate cell cycle arrest only at an early stage of follicle Ptenfl/fl;Amhr2-Cre mice (19), we next sought to determine if development when granulosa cells are proliferating. disrupting Pten in the Kras mutant genotypes would lead to tumor KrasG12D alters cell cycle and differentiation-related gene formation. Contrary to our hypothesis, double mutations of Pten expression profiles in granulosa cells. Gene expression profiling and Kras in granulosa cells of the KrasG12D;Amhr2-Cre and (Supplementary Table S1) and real-time reverse transcription-PCR KrasG12D;Cyp19-Cre mice failed to cause the development of GCTs (Fig. 3B) using total RNA from ovaries of KrasG12D;Amhr2-Cre mice but altered follicular growth, prevented ovulation, and rendered

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Figure 3. Pten/Kras double mutant mice exhibit signs of premature ovarian failure. Fertility of females in the various mouse strains was evaluated by super-ovulation and breeding assays (A). Kras and Pten/Kras double mutant mice have reduced numbers of ovarian follicles, high serum FSH, and lowserum estradiol, symptoms of premature ovarian failure (A). H&E staining of ovarian sections confirmed the altered ovarian phenotype of Pten/Kras mutant mice at 1 and 3 mo of age (A). Genes crucial for oocyte function and follicle development were down-regulated in Pten/Kras double mutant ovaries compared with wild-type at 3 mo of age (B). Cell cycle regulators were decreased in Pten/Kras double mutant ovaries (C). Cyclin A (red) and E2F1 (green) are highly expressed in granulosa cells present in growing follicles (arrows) in wild-type (WT) and Ptenfl/fl;KrasG12D;Cyp19-Cre mice, but not in cells present in abnormal follicles (asterisks) of 1-mo-old Ptenfl/fl;KrasG12D;Cyp19-Cre mice (D). the mice infertile (Fig. 3A), a phenotype more severe than that cells present in the abnormal follicle-like structures compared with of the KrasG12D;Amhr2-Cre and KrasG12D;Cyp19-Cre females (10). the normal-looking follicles in the 1-month-old Ptenfl/fl;KrasG12D; These results indicate that the Kras mutation plays a dominant Cyp19-Cre ovaries (Fig. 3D). role in granulosa cells and that the conditional knockout of Pten Double mutations of Pten and Kras in the Ptenfl/fl;KrasG12D; strengthened the effect of KrasG12D. Amhr2-Cre mice but not the Ptenfl/fl;KrasG12D;Cyp19-Cre mice Histopathologic examination of ovarian morphology indicated cause OSE cancer. Although GCTs did not develop in response to that the Ptenfl/fl;KrasG12D;Cyp19-Cre double-mutant mice showed double mutations in KrasG12D and Pten, the OSE was affected signs of premature ovarian failure. Ovaries from females older than selectively when recombination of both genes was driven by 3 months had reduced numbers of developing follicles (Fig. 3A), Amhr2-Cre. The Ptenfl/fl;KrasG12D;Amhr2-Cre mice developed ag- high levels of serum FSH, and low levels of serum estradiol gressive OSE tumors as early as 1 month of age (Fig. 4A and B, (Fig. 3A). Furthermore, granulosa cell marker genes and oocyte- Pten;Kras;A-Cre), whereas the Ptenfl/fl;KrasG12D;Cyp19-Cre females specific genes were dramatically decreased in Kras mutant ovaries did not (Fig. 4A and B; Pten;Kras;C-Cre; Supplementary Fig. S2). The (Supplementary Table S1; Fig. 3B). ovarian tumors present in the Ptenfl/fl;KrasG12D;Amhr2-Cre mice Western blot and immunofluorescent analyses show that cyclin have been classified as low-grade serous papillary cystadenocarci- D2, cyclin A, and E2F1 are highly expressed in granulosa cells of nomas by histologic criteria (Fig. 4; Supplementary Figs. S2 and S3) wild-type mice but are markedly reduced in ovaries of 3-month-old and by immunostaining of ovarian sections with the epithelial cell Ptenfl/fl;KrasG12D;Cyp19-Cre mice (Fig. 3C and D; data not shown). marker cytokeratin 8 (Fig. 4C), the OSE cell marker MUC 16, and Immunostaining of cyclin A and E2F1 was negligible in granulosa the serous adenocarcinoma cell marker WT-1 (Supplementary www.aacrjournals.org 6467 Cancer Res 2009; 69: (16). August 15, 2009

Figure 4. OSE cell cancers develop in the Ptenfl/fl;KrasG12D;Amhr2-Cre mice but not in the Ptenfl/fl;KrasG12D;Cyp19-Cre mice or Ptenfl/fl;KrasG12D;Pgr-Cre mice. Ptenfl/fl;KrasG12D;Amhr2-Cre female mice develop massive low-grade serous, papillary cystadenocarcinomas by 3 mo of age (A–C). Multifocally, the cystadenocarcinoma invades the ovarian parenchyma (B; top right) and surround the abnormal follicle-like structures (B; bottom left). Ovaries of the Ptenfl/fl; KrasG12D;Cyp19-Cre mice have larger ovaries than wild-type mice due to the hyperplasia of interstitial tissue, but do not develop ovarian tumors (A–C; right). The epithelial cell origin of tumors was confirmed by immunostaining of cytokeratin 8 in wild-type and Ptenfl/fl; KrasG12D;Cyp19-Cre mice (C; right) and in tumor structures invading the ovary (C; left) as well as in tumor-derived cysts (C; middle). LacZ-staining in Rosa26 reporter mouse strains showing localization to granulosa cells in Cyp19-Cre mice (D; middle) and to OSE cells in Amhr2-Cre mice (D; left). Ovaries from a 5-wk-old Ptenfl/fl;KrasG12D;Pgr-Cre mice have no abnormal follicle-like lesions and no OSE tumors (D; right), despite the development of massive uterine tumors (data not shown).

Fig. S3A–C). The development of OSE cell tumors in the Amhr2-Cre Cre mouse strain, ovaries of the Ptenfl/fl;KrasG12D;Pgr-Cre females mouse strain, but not the Cyp19-Cre strain, is caused by the showed relatively normal histology and were devoid of both the expression of Amhr2-Cre recombinase in OSE cells, as shown by follicle-derived ovarian lesions and OSE cell tumors (Fig. 4D, Ptenfl/fl; LacZ staining of OSE cells in the Rosa26;Amhr2-Cre but not in the KrasG12D;Pgr-Cre; right) despite the fact that they develop massive Rosa26;Cyp19-Cre reporter mouse strains (Fig. 4D). These results uterine tumors and die within 6 weeks after birth.4 extend those of Connolly and colleagues, who generated OSE tumors Due to the expansive growth of OSE cell tumors in the Ptenfl/fl; using the Amhr2 promoter to direct expression of the SV40 TAg (33), KrasG12D;Amhr2-Cre females, they die between 3 and 6 months of and Szotek and colleagues, who reported that cell lines derived from mouse ovarian epithelial cell tumors express AMHR2 (34). Furthermore, when the double mutations were made in the Pgr- 4 J. Wang and F. DeMayo, unpublished data.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Cell-Selective Effects of Kras and Pten in the Ovary age (Fig. 5A). In contrast, mice with single mutations of Pten Immunofluorescent staining of ovaries from 1-month-old Ptenfl/fl; (Ptenfl/fl;Amhr2-Cre) were viable (18) and rarely developed GCTs KrasG12D;Amhr2-Cre mice showed that AKT and its target kinases, (19)m whereas the KrasG12D;Amhr2-Cre and Ptenfl/fl;KrasG12D;Cyp19- mTOR and GSK3h, are highly phosphorylated in the invading OSE Cre femaleswereviablewithoutsignsofovariantumor tumor cells, but not in the nonmitotic granulosa cells (Fig. 5D). development (Fig. 5A), although some OSE cell hyperplasia was The OSE tumor cells showed active proliferation, based on the observed in the KrasG12D;Amhr2-Cre females at 10 months or incorporation of BrdU(Fig. 5 D) and immunostaining of prolifer- older (data not shown). The abnormal follicle-derived ovarian ating cell nuclear antigen (Fig. 5D). Strikingly, whereas expression lesions induced by KrasG12D were observed in the Pten/Kras of KRAS was silenced in granulosa cells of the KrasG12D-induced double mutant ovaries but the number decreased dramatically abnormal follicles (Fig. 2A), KRAS was high in OSE tumor cells and (Fig. 5B), suggesting that PTEN activity is required to maintain were associated with high levels of phosphorylated these structures. ERK1/2 and phosphorylated AKT but not phosphorylated p38 PI3K pathway is hyperactivated in Pten/KrasG12D-induced MAPK (Fig. 6A–C; Supplementary Fig. S3D). The inability of OSE OSE tumors. PTEN was expressed in the stromal cells and cells to inactivate RAS-related signaling events under oncogenic remnants of abnormal follicle-like structures present in the Ptenfl/fl; insults may explain why these cells, but not granulosa cells, are KrasG12D;Cyp19-Cre ovaries (Fig. 5B). By contrast, the OSE tumor susceptible to tumorigenic stimuli. cells present in the Ptenfl/fl;KrasG12D;Amhr2-Cre ovaries were completely devoid of PTEN (Fig. 5B) compared with the control OSE cells (Supplementary Fig. S4), and showed elevated activity of Discussion the PI3K pathway components compared with wild-type controls We document for the first time that the effects of expressing (Fig. 5C), presumably due to the increased AKT activity (Fig. 5D). KRASG12D in ovarian granulosa cells in vivo are dependent on the

Figure 5. The PI3K pathway is hyperactivated in Pten/Kras-induced OSE tumors. Conditional depletion of Pten in granulosa cells reduced the numbers of ovarian lesions in ovaries of both Ptenfl/fl;KrasG12D;Amhr2-Cre mice and Ptenfl/fl;KrasG12D;Cyp19-Cre mice (A). Most Ptenfl/fl;KrasG12D;Amhr2-Cre mice died of ovarian cancers between 3 and 6 mo of age (A). PTEN was expressed in the ovarian stroma of Ptenfl/fl;KrasG12D;Cyp19-Cre mice (B) but was absent in OSE tumor cells of Ptenfl/fl;KrasG12D;Amhr2-Cre mice (B). Western blot analyses (C) and immunofluorescent staining (D; left)of PI3K pathway components in OSE cells. BrdU is incorporated into proliferating granulosa cells in normal (WT) ovaries (D; top right) and the OSE tumor cells in Ptenfl/fl;KrasG12D;Amhr2-Cre mice (D; middle right). The OSE tumor cells also stained for proliferating cell nuclear antigens (PCNA; D; bottom right).

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Figure 6. KRAS-related signaling pathways are actively involved in the development of OSE cancers. KRAS is highly expressed by OSE tumor cells of Ptenfl/fl;KrasG12D;Amhr2-Cre mice (A) and this is associated with elevated levels of phosphorylated ERK1/2 (B) and phosphorylated AKT (C). The cell-specific and stage-specific effects of Kras/Pten mutations on OSE cells and granulosa cells were summarized schematically (D).

stage of granulosa cell differentiation. In proliferating granulosa transcriptional coregulators that mediate entry into the S phase cells present in small growing follicles of the KrasG12D;Amhr2-Cre (27). Because activin can affect granulosa cell proliferation (37, 38) mice and KrasG12D;Cyp19-Cre mice, the expression of KRASG12D led and has been linked to ovarian stromal tumors (39), it may be to cell cycle arrest and the absence of genes associated with a important that levels of Inhba and Inhbb are negligible in the differentiated granulosa cell phenotype. As a consequence, these KrasG12D mutant cells. That the small abnormal follicle-like mice become infertile. However, in the KrasG12D;Pgr-Cre mice, in structures persist and accumulate in the ovaries of the mutant which the expression of KRASG12D only occurs in differentiating mice is associated with, and may be dependent on, the lack of granulosa of preovulatory follicles after the LH/hCG surge, we apoptosis as well as cell cycle arrest (10). observed no overt effects on ovarian function and the mice The mechanisms regulating cell cycle arrest in the granulosa remained fertile. The consequences of depleting Pten in cells cells may be mediated, in part, by the silencing of the Kras gene, expressing KRASG12D were also cell type–specific. Whereas Kras/ thus impairing the phosphorylation of ERK1/2 and AKT. Although Pten mutant granulosa cells did not exhibit signs of increased KrasG12D is expressed at the time of recombination (10), the proliferation or transformation, OSE cells challenged with the same complete absence of KRAS protein in the mutant granulosa cells genetic alterations exhibited serous papillary cystadenocarcinomas suggests that KrasG12D silences the Kras gene. To our knowledge, within 1 month of age. These results document unequivocally that the silencing of the Kras gene by oncogenic KRASG12D has not been the effect of mutant KRASG12D and Pten depletion are cell type– reported previously, may represent an E2F1-mediated regulatory specific within the ovary and that granulosa cells, like some other mechanism (35), and seems to be independent of factors mitotic cells (31, 35), possess mechanisms to combat oncogenesis implicated in regulating cell cycle arrest and senescence in other by entering cell cycle arrest (Fig. 6D). cell types (refs. 31, 40, 41; Supplementary Figs. S1 and S2). That Specifically, our results document in six in vivo mutant mouse silencing the Kras gene is critical for preventing GCT formation is À models that KRASG12D potently affects the proliferative capacity of supported by the observations that the Ptenfl/fl;Ctnnb1exon3fl/ ; granulosa cells. If recombination occurs in granulosa cells of small Amhr2-Cre mice expressing constitutively active h-catenin growing follicles, these cells become nonmitotic (10) and express (CTNNB1) in the Pten-null background develop metastatic GCTs extremely low levels of genes and proteins related to cell cycle with 100% penetrance at an early age (19). Thus, the response of progression, especially factors regulating entry into S phase of the granulosa cells to oncogenic factors depends on which combina- cell cycle (27, 36) including cyclin A, cyclin D2, and E2F1. Whereas tions of oncogenic stimuli are expressed in these cells. cyclin A and cyclin D2 regulate specific cell cycle–dependent Additional mechanisms controlling cell cycle arrest in the Kras kinases, E2F1 regulates the transcription of cyclin A and other mutant granulosa cells seem to be associated with and perhaps

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Cell-Selective Effects of Kras and Pten in the Ovary mediated by elevated levels of the tumor suppressor PTEN, levels of phosphorylated p38 MAPK, whereas the mutant OSE did phosphorylated p38 MAPK-mediated events, and/or the repressor not. Thus, these results, combined with those of Connolly and of transcription Zbtb16 (Plzf ), all of which are increased in the colleagues (33) and Szotek and colleagues (48), clearly document KrasG12D mutant cells. Because PLZF is a potent regulator of that the Amhr2 gene is expressed in normal OSE cells. Although its germ cell stem cell survival (28, 42) and has been linked to cell role in normal, nontransformed cells remains to be clarified, AMH senescence (43), it may participate in the extended life span and has been shown to block the proliferation of mouse and human lack of differentiation of these somatic granulosa cells. That ovarian cancer cell lines (48). Thus, the spontaneous and cell-specific phosphorylated p38 MAPK is elevated markedly in the nonmitotic development of OSE cell tumors in the ovaries of the Ptenfl/fl, granulosa cells may be important because phosphorylated p38 KrasG12D;Amhr2Cre mice described herein provides the first MAPK is associated with senescent fibroblasts and epithelial cells demonstration that Amhr2-Cre is expressed in murine OSE cells (29, 30). Of note, the highly proliferative OSE tumor cells did not and can drive oncogenic transformation by generating cell-specific exhibit detectable levels of phosphorylated p38 MAPK. The mutant mutants. Our results confirm and extend the studies of others granulosa cells within the abnormal follicle-like structures also who have generated OSE cell tumors by injecting adenoviral-Cre express markedly elevated levels of PTEN and negligible levels of vectors into the ovarian bursa of Ptenfl/fl;KrasG12D mice (49) and phosphorylated AKT, indicating that the PI3K pathway is severely Apcfl/fl;Ptenfl/fl mice (50). Furthermore, our results document that the inhibited. Although we hypothesized that disrupting Pten in the OSE cells in Ptenfl/fl,KrasG12D;Amhr2-Cre mice develop tumors KrasG12D mutant strains might prevent cell cycle arrest and lead to whereas the granulosa cells do not. GCT formation, the loss of Pten in the KrasG12D-expressing cells did In summary, these studies document that expression of KrasG12D not prevent the formation of abnormal follicle-like structures. and disruption of the Pten gene in granulosa cells results in a However, the number of abnormal follicle-like structures was phenotype distinct from that occurring in OSE cells present in the reduced, and in some ovaries of the double Kras/Pten mutant mice, same ovary (Fig. 6D). Whereas expression of KRASG12D leads to cell regions of the ovary contained luteinized-like structures. Because cycle arrest in granulosa cells in which the Kras gene is silenced, it this ovarian phenotype is more reminiscent of that observed in the promotes proliferation in OSE cells in which KRAS remains Pten conditional knockout ovaries (18), it is possible that if the elevated. When Pten is disrupted in the KrasG12D strain, it enhances disruption of Pten occurs simultaneously with the onset of OSE cell proliferation and tumor formation, at least in part, by the KrasG12D expression, the granulosa cells may luteinize. This is sustained phosphorylation and activation of ERK1/2 and PI3K likely because KrasG12D expression did not affect ovarian cell pathway components and perhaps the suppression of p38 MAPK function if expressed in luteinizing granulosa cells of the Ptenfl/fl; activation. Conversely, disrupting Pten in the Kras mutant KrasG12D;PGR-Cre mice. This agrees with previous reports indi- granulosa cells does not reverse cell cycle arrest or induce cating that KrasG12D can cause cell cycle arrest and senescence tumorigenesis of these cells (Fig. 6D). By understanding how only in mitotic cells (44), and that the RAS-ERK1/2 pathway is KRASG12D can exert completely opposite effects in these two cell critical for cell cycle arrest during luteinization (45). Remarkably, populations within the same tissue may provide a key for no GCTs formed in the ovaries of Ptenfl/fl,KrasG12D;Cyp19-Cre mice controlling cancer development and progression. One key may be even at 10 months of age (data not shown), and were rare in the the total silencing of the Kras gene itself. Pten fl/fl;Amhr2-Cre mice (19). In marked contrast to the granulosa cells, the OSE cells present Disclosure of Potential Conflicts of Interest in the Ptenfl/fl;KrasG12D;Amhr2-Cre mouse ovaries transformed rapidly and developed into serous papillary cystadenocarcinomas No potential conflicts of interest were disclosed. between 1 and 3 months of age. That the tumors are derived from OSE cells confirmed several criteria: LacZ staining of OSE cells only Acknowledgments in Amhr2-Cre mice and the presence of the epithelial marker Received 8/28/08; revised 5/14/09; accepted 6/11/09. cytokeratin 8, an OSE cell marker that has been detected recently Grant support: NIH grants NIH-HD16229, NIH-HD16272, NIH-HD07494; a in the apical cell membrane of mouse serous adenocarcinoma of Specialized Cooperative Centers Program in Reproduction and Infertility Research, Project II (J.S. Richards), a Postdoctoral Fellowship HD-07165 (H.-Y. Fan), 1 U01 the ovary and human ovarian carcinoma, MUC16 (46, 47), and a CA105352 (F. DeMayo), and NIH/NCI CA-077530 (J.P. Lydon). specific marker of human ovarian serous papillary adenocarcino- 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 mas, WT-1. The Kras/Pten mutant OSE cells expressed elevated with 18 U.S.C. Section 1734 solely to indicate this fact. levels of PI3K pathway components and phosphorylated ERK1/2, We thank Drs. Jan Gossen, Tyler Jacks, Richard Behringer, and Hong Wu for all of which were localized selectively to the invading OSE cells providing the Cyp19-Cre mice, LSL-KrasG12D mice, Amhr2-Cre mice, and Ptenfl/fl mice, respectively. We also thank Drs. Richard Behringer and Robert Bast, Jr., for the MUC16 and were not present in the abnormal follicle-like structures. antibody, Dr. Michael Mancini and members of the Microscopy Core (NIH-HD-07495) Conversely, the abnormal follicle-like structures contained high for their time and assistance, and Yuet Lo for her many contributions.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Cell Type−Specific Targeted Mutations of Kras and Pten Document Proliferation Arrest in Granulosa Cells versus Oncogenic Insult to Ovarian Surface Epithelial Cells

Heng-Yu Fan, Zhilin Liu, Marilene Paquet, et al.

Cancer Res 2009;69:6463-6472.

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