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Mammalian target of rapamycin is a therapeutic target for murine ovarian endometrioid adenocarcinomas with dysregulated Wnt/β-catenin and PTEN.

Tanwar PS, Zhang L, Kaneko-Tarui T, Curley MD, Taketo MM, Rani P, Roberts DJ, Teixeira JM - PLoS ONE (2011)

Bottom Line: Mutations in the WNT and PI3K pathways are frequently observed in the human ovarian endometrioid adenocarcinomas (OEAs).However, the role of WNT/β-catenin and PTEN/AKT signaling in the etiology and/or progression of this disease is currently unclear.These studies demonstrate that rapamycin might be an effective therapeutic for human ovarian endometrioid patients with dysregulated Wnt/β-catenin and Pten/PI3K signaling.

View Article: PubMed Central - PubMed

Affiliation: Vincent Center for Reproductive Biology, Department of Obstetrics, Gynecology, and Reproductive Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Despite the fact that epithelial ovarian cancers are the leading cause of death from gynecological cancer, very little is known about the pathophysiology of the disease. Mutations in the WNT and PI3K pathways are frequently observed in the human ovarian endometrioid adenocarcinomas (OEAs). However, the role of WNT/β-catenin and PTEN/AKT signaling in the etiology and/or progression of this disease is currently unclear. In this report we show that mice with a gain-of-function mutation in β-catenin that leads to dysregulated nuclear accumulation of β-catenin expression in the ovarian surface epithelium (OSE) cells develop indolent, undifferentiated tumors with both mesenchymal and epithelial characteristics. Combining dysregulated β-catenin with homozygous deletion of PTEN in the OSE resulted in development of significantly more aggressive tumors, which was correlated with inhibition of p53 expression and cellular senescence. Induced expression of both mTOR kinase, a master regulator of proliferation, and phosphorylation of its downstream target, S6Kinase was also observed in both the indolent and aggressive mouse tumors, as well as in human OEA with nuclear β-catenin accumulation. Ectopic allotransplants of the mouse ovarian tumor cells with a gain-of-function mutation in β-catenin and PTEN deletion developed into tumors with OEA histology, the growth of which were significantly inhibited by oral rapamycin treatment. These studies demonstrate that rapamycin might be an effective therapeutic for human ovarian endometrioid patients with dysregulated Wnt/β-catenin and Pten/PI3K signaling.

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Overactive Wnt/β-catenin in human OEAs and mutant Amhr2-Cre;Ctnnb1Δ(ex3)/+ mice.(A) Membranous β-catenin expression in normal postmenopausal human ovarian surface epithelium (OSE) was detected by immunofluorescence. (B–D) Nuclear β-catenin in representative tissue samples obtained from three different human OEA patients. β-catenin protein expression in control Ctnnb1Δ(ex3)/+ (E) and in Amhr2-Cre;Ctnnb1Δ(ex3)/+ (F) ovaries of 4-week-old mice. Insert in Panels E and F are higher magnification images of follicles to show membranous β-catenin expression in granulosa cells. (G) Higher magnification image of area indicated with a white arrowhead in panel F. (H) Western blot analyses of β-catenin in protein lysate obtained from granulosa cells, remnant (remaining ovarian tissue after granulosa cell isolation including ovarian surface epithelium), whole control (Ctnnb1Δ(ex3)/+) and mutant (Amhr2-Cre;Ctnnb1Δ(ex3)/+) 4-week-old ovaries, tumors from the mutant (Amhr2-Cre;Ctnnb1Δ(ex3)/+) 6-month-old ovaries. White arrows in A, E, F, and G mark the ovarian surface epithelial cells. Nuclei are stained with DAPI. Bars = 50 um.
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pone-0020715-g001: Overactive Wnt/β-catenin in human OEAs and mutant Amhr2-Cre;Ctnnb1Δ(ex3)/+ mice.(A) Membranous β-catenin expression in normal postmenopausal human ovarian surface epithelium (OSE) was detected by immunofluorescence. (B–D) Nuclear β-catenin in representative tissue samples obtained from three different human OEA patients. β-catenin protein expression in control Ctnnb1Δ(ex3)/+ (E) and in Amhr2-Cre;Ctnnb1Δ(ex3)/+ (F) ovaries of 4-week-old mice. Insert in Panels E and F are higher magnification images of follicles to show membranous β-catenin expression in granulosa cells. (G) Higher magnification image of area indicated with a white arrowhead in panel F. (H) Western blot analyses of β-catenin in protein lysate obtained from granulosa cells, remnant (remaining ovarian tissue after granulosa cell isolation including ovarian surface epithelium), whole control (Ctnnb1Δ(ex3)/+) and mutant (Amhr2-Cre;Ctnnb1Δ(ex3)/+) 4-week-old ovaries, tumors from the mutant (Amhr2-Cre;Ctnnb1Δ(ex3)/+) 6-month-old ovaries. White arrows in A, E, F, and G mark the ovarian surface epithelial cells. Nuclei are stained with DAPI. Bars = 50 um.

Mentions: Because mutations in Wnt/β-catenin signaling components are frequently observed in human OEAs patients [11], [13], we examined β-catenin protein expression in human OEA tissue samples and observed nuclear accumulation of β-catenin, which is indicative of Wnt pathway activation, in 80% (4/5) (Fig. 1B–D). Only membranous β-catenin protein expression was observed in OSE of normal human ovary (Fig. 1A). To confirm that activated Wnt/β-catenin signaling can initiate the formation of OEAs, we developed a β-catenin gain-of-function mouse model by conditionally deleting exon 3 (Ex3) of the β-catenin gene (Ctnnb1), which contains the phosphorylation sites of β-catenin required for its ubiquitination and subsequent degradation [12]. Deletion of exon 3 leads to formation of a degradation-resistant but functional form of β-catenin, which accumulates in the cytoplasm and nuclei of cells [12]. Amhr2-cre mice were used to mate with mice with a flox allele of β-catenin, Ctnnb1fl(ex3)/+ because Amhr2 is expressed in OSE and Amhr2 promoter-driven Cre causes efficient recombination in OSE cells [2], [19], [22], [23], [24]. In order to verify whether activation of β-catenin occurs in OSE cells of Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries, we analyzed expression of β-catenin. Examination of four-week-old mice showed nuclear accumulation of β-catenin in OSE and OSE-derived lesions present throughout the cortex of the mutant Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries (Fig. 1F & G)). In contrast, only membranous β-catenin expression was observed in the OSE of age matched control ovaries (Fig. 1E). Membranous β-catenin expression was also observed in the granulosa cells of both mutant and control Ctnnb1fl(ex3)/+ ovaries (Fig. 1E & F, inserts). We next performed western blot analyses on ovarian lysates and demonstrated the presence of full length and exon 3-deleted β-catenin in the granulosa cell-depleted residual ovarian cells, including those from OSE and tumor in the mutant animals (Fig. 1H). A relatively weaker band of exon 3-deleted β-catenin was detected in the granulosa cells of the mutant ovaries indicating that very low recombination occurred in granulosa cells, which is consistent with the largely membranous staining observed in Fig. 1F or that the granulosa cell preparations were contaminated with ovarian stromal cells.


Mammalian target of rapamycin is a therapeutic target for murine ovarian endometrioid adenocarcinomas with dysregulated Wnt/β-catenin and PTEN.

Tanwar PS, Zhang L, Kaneko-Tarui T, Curley MD, Taketo MM, Rani P, Roberts DJ, Teixeira JM - PLoS ONE (2011)

Overactive Wnt/β-catenin in human OEAs and mutant Amhr2-Cre;Ctnnb1Δ(ex3)/+ mice.(A) Membranous β-catenin expression in normal postmenopausal human ovarian surface epithelium (OSE) was detected by immunofluorescence. (B–D) Nuclear β-catenin in representative tissue samples obtained from three different human OEA patients. β-catenin protein expression in control Ctnnb1Δ(ex3)/+ (E) and in Amhr2-Cre;Ctnnb1Δ(ex3)/+ (F) ovaries of 4-week-old mice. Insert in Panels E and F are higher magnification images of follicles to show membranous β-catenin expression in granulosa cells. (G) Higher magnification image of area indicated with a white arrowhead in panel F. (H) Western blot analyses of β-catenin in protein lysate obtained from granulosa cells, remnant (remaining ovarian tissue after granulosa cell isolation including ovarian surface epithelium), whole control (Ctnnb1Δ(ex3)/+) and mutant (Amhr2-Cre;Ctnnb1Δ(ex3)/+) 4-week-old ovaries, tumors from the mutant (Amhr2-Cre;Ctnnb1Δ(ex3)/+) 6-month-old ovaries. White arrows in A, E, F, and G mark the ovarian surface epithelial cells. Nuclei are stained with DAPI. Bars = 50 um.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111436&req=5

pone-0020715-g001: Overactive Wnt/β-catenin in human OEAs and mutant Amhr2-Cre;Ctnnb1Δ(ex3)/+ mice.(A) Membranous β-catenin expression in normal postmenopausal human ovarian surface epithelium (OSE) was detected by immunofluorescence. (B–D) Nuclear β-catenin in representative tissue samples obtained from three different human OEA patients. β-catenin protein expression in control Ctnnb1Δ(ex3)/+ (E) and in Amhr2-Cre;Ctnnb1Δ(ex3)/+ (F) ovaries of 4-week-old mice. Insert in Panels E and F are higher magnification images of follicles to show membranous β-catenin expression in granulosa cells. (G) Higher magnification image of area indicated with a white arrowhead in panel F. (H) Western blot analyses of β-catenin in protein lysate obtained from granulosa cells, remnant (remaining ovarian tissue after granulosa cell isolation including ovarian surface epithelium), whole control (Ctnnb1Δ(ex3)/+) and mutant (Amhr2-Cre;Ctnnb1Δ(ex3)/+) 4-week-old ovaries, tumors from the mutant (Amhr2-Cre;Ctnnb1Δ(ex3)/+) 6-month-old ovaries. White arrows in A, E, F, and G mark the ovarian surface epithelial cells. Nuclei are stained with DAPI. Bars = 50 um.
Mentions: Because mutations in Wnt/β-catenin signaling components are frequently observed in human OEAs patients [11], [13], we examined β-catenin protein expression in human OEA tissue samples and observed nuclear accumulation of β-catenin, which is indicative of Wnt pathway activation, in 80% (4/5) (Fig. 1B–D). Only membranous β-catenin protein expression was observed in OSE of normal human ovary (Fig. 1A). To confirm that activated Wnt/β-catenin signaling can initiate the formation of OEAs, we developed a β-catenin gain-of-function mouse model by conditionally deleting exon 3 (Ex3) of the β-catenin gene (Ctnnb1), which contains the phosphorylation sites of β-catenin required for its ubiquitination and subsequent degradation [12]. Deletion of exon 3 leads to formation of a degradation-resistant but functional form of β-catenin, which accumulates in the cytoplasm and nuclei of cells [12]. Amhr2-cre mice were used to mate with mice with a flox allele of β-catenin, Ctnnb1fl(ex3)/+ because Amhr2 is expressed in OSE and Amhr2 promoter-driven Cre causes efficient recombination in OSE cells [2], [19], [22], [23], [24]. In order to verify whether activation of β-catenin occurs in OSE cells of Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries, we analyzed expression of β-catenin. Examination of four-week-old mice showed nuclear accumulation of β-catenin in OSE and OSE-derived lesions present throughout the cortex of the mutant Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries (Fig. 1F & G)). In contrast, only membranous β-catenin expression was observed in the OSE of age matched control ovaries (Fig. 1E). Membranous β-catenin expression was also observed in the granulosa cells of both mutant and control Ctnnb1fl(ex3)/+ ovaries (Fig. 1E & F, inserts). We next performed western blot analyses on ovarian lysates and demonstrated the presence of full length and exon 3-deleted β-catenin in the granulosa cell-depleted residual ovarian cells, including those from OSE and tumor in the mutant animals (Fig. 1H). A relatively weaker band of exon 3-deleted β-catenin was detected in the granulosa cells of the mutant ovaries indicating that very low recombination occurred in granulosa cells, which is consistent with the largely membranous staining observed in Fig. 1F or that the granulosa cell preparations were contaminated with ovarian stromal cells.

Bottom Line: Mutations in the WNT and PI3K pathways are frequently observed in the human ovarian endometrioid adenocarcinomas (OEAs).However, the role of WNT/β-catenin and PTEN/AKT signaling in the etiology and/or progression of this disease is currently unclear.These studies demonstrate that rapamycin might be an effective therapeutic for human ovarian endometrioid patients with dysregulated Wnt/β-catenin and Pten/PI3K signaling.

View Article: PubMed Central - PubMed

Affiliation: Vincent Center for Reproductive Biology, Department of Obstetrics, Gynecology, and Reproductive Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Despite the fact that epithelial ovarian cancers are the leading cause of death from gynecological cancer, very little is known about the pathophysiology of the disease. Mutations in the WNT and PI3K pathways are frequently observed in the human ovarian endometrioid adenocarcinomas (OEAs). However, the role of WNT/β-catenin and PTEN/AKT signaling in the etiology and/or progression of this disease is currently unclear. In this report we show that mice with a gain-of-function mutation in β-catenin that leads to dysregulated nuclear accumulation of β-catenin expression in the ovarian surface epithelium (OSE) cells develop indolent, undifferentiated tumors with both mesenchymal and epithelial characteristics. Combining dysregulated β-catenin with homozygous deletion of PTEN in the OSE resulted in development of significantly more aggressive tumors, which was correlated with inhibition of p53 expression and cellular senescence. Induced expression of both mTOR kinase, a master regulator of proliferation, and phosphorylation of its downstream target, S6Kinase was also observed in both the indolent and aggressive mouse tumors, as well as in human OEA with nuclear β-catenin accumulation. Ectopic allotransplants of the mouse ovarian tumor cells with a gain-of-function mutation in β-catenin and PTEN deletion developed into tumors with OEA histology, the growth of which were significantly inhibited by oral rapamycin treatment. These studies demonstrate that rapamycin might be an effective therapeutic for human ovarian endometrioid patients with dysregulated Wnt/β-catenin and Pten/PI3K signaling.

Show MeSH
Related in: MedlinePlus