Synergistic effects of Pten loss and WNT/CTNNB1 signaling pathway activation in ovarian granulosa cell tumor development and progression

Abstract

The mechanisms of granulosa cell tumor (GCT) development may involve the dysregulation of signaling pathways downstream of follicle-stimulating hormone, including the phosphoinosite-3 kinase (PI3K)/AKT pathway. To test this hypothesis, a genetically engineered mouse model was created to derepress the PI3K/AKT pathway in granulosa cells by conditional targeting of the PI3K antagonist gene Pten (Pten(flox/flox);Amhr2(cre/+)). The majority of Pten(flox/flox);Amhr2(cre/+) mice featured no ovarian anomalies, but occasionally ( approximately 7%) developed aggressive, anaplastic GCT with pulmonary metastases. The expression of the PI3K/AKT downstream effector FOXO1 was abrogated in Pten(flox/flox);Amhr2(cre/+) GCT, indicating a mechanism by which GCT cells may increase proliferation and evade apoptosis. To relate these findings to spontaneously occurring GCT, analyses of PTEN and phospho-AKT expression were performed on human and equine tumors. Although PTEN loss was not detected, many GCT (2/5 human, 7/17 equine) featured abnormal nuclear or perinuclear localization of phospho-AKT, suggestive of altered PI3K/AKT activity. As inappropriate activation of WNT/CTNNB1 signaling causes late-onset GCT development and cross talk between the PI3K/AKT and WNT/CTNNB1 pathways has been reported, we tested whether these pathways could synergize in GCT. Activation of both the PI3K/AKT and WNT/CTNNB1 pathways in the granulosa cells of a mouse model (Pten(flox/flox);Ctnnb1(flox(ex3)/+);Amhr2(cre/+)) resulted in the development of GCT similar to those observed in Pten(flox/flox);Amhr2(cre/+) mice, but with 100% penetrance, perinatal onset, extremely rapid growth and the ability to spread by seeding into the abdominal cavity. These data indicate a synergistic effect of dysregulated PI3K/AKT and WNT/CTNNB1 signaling in the development and progression of GCT and provide the first animal models for metastatic GCT.

Fig. 1.

Schematic representation of cross talk between PKA, PI3K/AKT and WNT/CTNNB1 intracellular signaling pathways. AC = adenylyl cyclase, APC = adenomatosis polyposis coli, CREB = cAMP response element binding, DSH = disheveled, FOXO1 = forkhead box O1, FSH(R) = follicle stimulating hormone (receptor), FZD = frizzled, IGF1(R) = insulin-like growth factor-1 (receptor), LRP = low-density lipoprotein receptor-related protein, PDK = 3-phosphoinositide-dependent protein kinase, PI3K = phosphoinositide 3-kinase, PKA = protein kinase A, PTEN = phosphatase and tensin homolog, SFK = SRC family protein kinases, TCF/LEF = transcription factor, T-cell specific/lymphoid enhancer factor 1 and CTNNB1 = β-catenin.

Fig. 2.

Lack of ovarian anomalies in most Pten^flox/flox;Amhr2^cre/+ mice. (A) Histological sections of ovaries from 5-month-old Pten^flox/flox and Pten^flox/flox;Amhr2^cre/+ mice. Original magnification, ×50. (B) PCR analysis of Cre-mediated recombination of the floxed Pten allele. Ovarian DNA was isolated from 1-month-old Pten^flox/flox and Pten^flox/flox;Amhr2^cre/+ mice (second through seventh lanes, inclusively). Positive controls for the floxed allele (Floxed CTRL) and wild-type allele (WT CTRL) were DNA from tail biopsies taken from a Pten^flox/flox and a wild-type mouse, respectively. Granulosa cells tumor DNA was isolated from 10-week-old Pten^flox/flox;Amhr2^cre/+ females (last two lanes). Expected PCR product sizes for the floxed, wild-type (WT) and cre-recombined (KO) alleles are indicated on the left. An additional ∼500 bp PCR product was commonly observed in samples of all genotypes and presumed to be an artifact unrelated to Pten. MW = molecular weight standards. (C) Immunoblot analyses of PTEN expression in isolated Pten^flox/flox and Pten^flox/flox;Amhr2^cre/+ granulosa cells. Each lane represents a distinct protein sample derived from the pooled granulosa cells from three non-tumor bearing mice. Actin was used as a loading control. (D) Immunohistochemical analysis of phospho-AKT expression in Pten^flox/flox and Pten^flox/flox;Amhr2^cre/+ ovaries. O = oocyte. Original magnification, ×1000.

Fig. 3.

Rare Pten^flox/flox;Amhr2^cre/+ mice develop aggressive, metastatic GCT. (A) Abdominal cavity of a 10-week-old Pten^flox/flox;Amhr2^cre/+ mouse bearing two large GCT. (B and C) Microscopic views of solid (B) and trabecular (C) GCT histological patterns. Original magnifications, 100× (B) or 200× (C). (D) Photomicrograph showing distinct cell populations within Pten^flox/flox;Amhr2^cre/+ GCT, characterized by higher (HA) or lesser (LA) degrees of anaplasia. Original magnification, ×400. (E) Microscopic view of a focus of ossification (O) within a Pten^flox/flox;Amhr2^cre/+ GCT. Original magnification, ×200. (F) Photomicrograph showing the presence of tumor cell embolisms in the lungs of the animal shown in (A). The arrow indicates an example of an embolism (i.e. the tumor cells are entirely contained within a vascular structure). Original magnification, ×200. (G) Photomicrograph showing the presence of GCT metastases (arrows) in the lungs of the animal shown in (A). Original magnification, ×400. (H) Photomicrographs comparing the highly anaplastic cells from the tumors (GCT) to those in the pulmonary metastases (PM). Original magnification, ×1000.

Fig. 4.

Altered PI3K/AKT signaling in Pten^flox/flox;Amhr2^cre/+ GCT. Immunoblot analyses of the indicated PI3K/AKT pathway signaling effectors in Pten^flox/flox granulosa cells (first two lanes) and Pten^flox/flox;Amhr2^cre/+ granulosa cells tumors (final four lanes). Actin was used as a loading control.

Fig. 5.

Abnormal PI3K/AKT pathway activity occurs in many GCT. (A) Immunohistochemical analysis of phospho-AKT expression in human and equine normal ovaries and granulosa cells tumors. Arrows indicate examples of cells showing perinuclear (image c) or nuclear (image d) staining. Original magnification, ×1000. (B) Immunohistochemical analysis of PTEN expression in human normal ovaries and granulosa cells tumors. Original magnification, ×200. Arrows indicate staining in non-tumoral stromal cells, little or no staining was found in the tumor cells. (C) Semiquantitative reverse transcription–PCR analysis of PTEN expression in human normal ovaries, granulosa cells tumors and isolated luteinizing granulosa cells (GC). GAPDH was used as a control gene.

Fig. 6.

Pten^flox/flox;Ctnnb1^flox(ex3)/+;Amhr2^cre/+ mice develop early-onset GCT with a high penetrance. (A) A 6-week-old Pten^flox/flox;Ctnnb1^flox(ex3)/+;Amhr2^cre/+ mouse bearing two large GCT. (B) Photomicrographs showing distinct cell populations within Pten^flox/flox;Ctnnb1^flox(ex3)/+;Amhr2^cre/+ GCT, characterized by higher (HA) or lesser (LA) degrees of anaplasia. (C) Photomicrograph of a newborn Pten^flox/flox;Ctnnb1^flox(ex3)/+;Amhr2^cre/+ ovary. Growing nests of dysplastic cells are circumscribed with dotted lines. Original magnification, ×400. (D) Photomicrograph of a pulmonary tumor cell embolism (arrow) from a 6-week-old female Pten^flox/flox;Ctnnb1^flox(ex3)/+;Amhr2^cre/+ mouse. Original magnification, ×400. (E–H) Photomicrographs depicting GCT pulmonary metastases (panel E, arrows), GCT cell invasion of the abdominal wall (panel F, M = muscular layer of the abdominal wall, arrows indicate invading GCT cells), a liver GCT metastasis (panel G, L = liver) and an adrenal GCT metastasis (panel H, M = adrenal medulla, C = adrenal cortex) from Pten^flox/flox;Ctnnb1^flox(ex3)/+;Amhr2^cre/+ mice killed several weeks after surgical removal of the primary ovarian tumors. Original magnification, ×40 (E) or ×200 (F–H).