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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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Sustained activation of β-catenin in the somatic cells leads to the induction of p53-mediated senescence in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries.(Panel A–F) Colocalization of β-catenin and PTEN in serial sections of control (Ctnnb1Δ(ex3)/+), Amhr2-Cre;Ctnnb1Δ(ex3)/+ and Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. Insets in Panels B, E, & F are higher magnification images of areas marked by black rectangles. F: follicle, t: tumor. (Panel G & H) Gross analyses of the reproductive tracts from the indicated mice show large tumors by 6 weeks in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries but relatively normal size ovaries in Amhr2-Cre;Ctnnb1Δ(ex3)/+ mice . SAβ-gal staining was observed in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries but not in control or Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries (I–K). Compared to controls and Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries, very few pH3 positive cells were present in precancerous lesions (white dotted lines) of Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries (L–N). Absence of TUNEL staining in pretumoral lesions (white dotted lines) showed that non-proliferating cells in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries are not apoptotic (O–Q). Immunohistochemical analyses showed an increase in p53 accumulation in Amhr2-Cre;Ctnnb1Δ(ex3)/+ tumors compared to controls and loss of p53 expression in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ tumors (R–T). Evidence of DNA damage determined by staining for γ-H2AX and observed in the tumors of both mutants (V & W) but not in the control ovaries (U). The differential expression of p53 was confirmed by Western blot analyses with pooled ovarian lysates (n = 4) and repeated three times (Panel X), which also confirmed the presence of exon 3 deleted β-catenin (arrowhead) in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. Analyses also showed increased expression of pAKT and pS6K, and decreased expression of p21 and p27kip1 in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. β-actin was used as a loading control. Bars = 50 um.
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pone-0020715-g003: Sustained activation of β-catenin in the somatic cells leads to the induction of p53-mediated senescence in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries.(Panel A–F) Colocalization of β-catenin and PTEN in serial sections of control (Ctnnb1Δ(ex3)/+), Amhr2-Cre;Ctnnb1Δ(ex3)/+ and Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. Insets in Panels B, E, & F are higher magnification images of areas marked by black rectangles. F: follicle, t: tumor. (Panel G & H) Gross analyses of the reproductive tracts from the indicated mice show large tumors by 6 weeks in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries but relatively normal size ovaries in Amhr2-Cre;Ctnnb1Δ(ex3)/+ mice . SAβ-gal staining was observed in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries but not in control or Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries (I–K). Compared to controls and Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries, very few pH3 positive cells were present in precancerous lesions (white dotted lines) of Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries (L–N). Absence of TUNEL staining in pretumoral lesions (white dotted lines) showed that non-proliferating cells in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries are not apoptotic (O–Q). Immunohistochemical analyses showed an increase in p53 accumulation in Amhr2-Cre;Ctnnb1Δ(ex3)/+ tumors compared to controls and loss of p53 expression in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ tumors (R–T). Evidence of DNA damage determined by staining for γ-H2AX and observed in the tumors of both mutants (V & W) but not in the control ovaries (U). The differential expression of p53 was confirmed by Western blot analyses with pooled ovarian lysates (n = 4) and repeated three times (Panel X), which also confirmed the presence of exon 3 deleted β-catenin (arrowhead) in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. Analyses also showed increased expression of pAKT and pS6K, and decreased expression of p21 and p27kip1 in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. β-actin was used as a loading control. Bars = 50 um.

Mentions: Interactions between Wnt/β-catenin and Akt/PTEN signaling pathways play an important role in carcinogenesis [6]. We examined the status of PTEN by performing immunohistochemical staining for β-catenin and PTEN on serial sections of Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries and observed increased expression of PTEN in pretumoral lesions with nuclear β-catenin (Fig. 3B & E). To examine whether PTEN deletion could affect tumor progression in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries, we developed another mouse model by deleting exon 3 of the β-catenin in PTEN negative cells of the mouse ovary (Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ) (Fig. 3C & F). These mice showed early onset of tumor development and were euthanized because of tumor-related morbidities (Fig. 3G & H). No evidence of ascites or metastases was observed by gross examination of the peritoneal cavity. The ovarian tumors in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries showed histopathological features similar to the tumors of Amhr2-Cre;Ctnnb1Δ(ex3)/+ mice (Fig. S2). Consistent with previous observations [22], [30], deletion of PTEN alone does not result in ovarian tumor development (data not shown), suggesting that activation of the phosphatidylinositol-3′ kinase (PI3K) signaling pathway with inactivating PTEN mutations alone may not be sufficient for tumor initiation and needs to act in concert with other oncogenes to cause cancer.


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)

Sustained activation of β-catenin in the somatic cells leads to the induction of p53-mediated senescence in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries.(Panel A–F) Colocalization of β-catenin and PTEN in serial sections of control (Ctnnb1Δ(ex3)/+), Amhr2-Cre;Ctnnb1Δ(ex3)/+ and Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. Insets in Panels B, E, & F are higher magnification images of areas marked by black rectangles. F: follicle, t: tumor. (Panel G & H) Gross analyses of the reproductive tracts from the indicated mice show large tumors by 6 weeks in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries but relatively normal size ovaries in Amhr2-Cre;Ctnnb1Δ(ex3)/+ mice . SAβ-gal staining was observed in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries but not in control or Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries (I–K). Compared to controls and Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries, very few pH3 positive cells were present in precancerous lesions (white dotted lines) of Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries (L–N). Absence of TUNEL staining in pretumoral lesions (white dotted lines) showed that non-proliferating cells in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries are not apoptotic (O–Q). Immunohistochemical analyses showed an increase in p53 accumulation in Amhr2-Cre;Ctnnb1Δ(ex3)/+ tumors compared to controls and loss of p53 expression in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ tumors (R–T). Evidence of DNA damage determined by staining for γ-H2AX and observed in the tumors of both mutants (V & W) but not in the control ovaries (U). The differential expression of p53 was confirmed by Western blot analyses with pooled ovarian lysates (n = 4) and repeated three times (Panel X), which also confirmed the presence of exon 3 deleted β-catenin (arrowhead) in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. Analyses also showed increased expression of pAKT and pS6K, and decreased expression of p21 and p27kip1 in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. β-actin was used as a loading control. Bars = 50 um.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111436&req=5

pone-0020715-g003: Sustained activation of β-catenin in the somatic cells leads to the induction of p53-mediated senescence in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries.(Panel A–F) Colocalization of β-catenin and PTEN in serial sections of control (Ctnnb1Δ(ex3)/+), Amhr2-Cre;Ctnnb1Δ(ex3)/+ and Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. Insets in Panels B, E, & F are higher magnification images of areas marked by black rectangles. F: follicle, t: tumor. (Panel G & H) Gross analyses of the reproductive tracts from the indicated mice show large tumors by 6 weeks in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries but relatively normal size ovaries in Amhr2-Cre;Ctnnb1Δ(ex3)/+ mice . SAβ-gal staining was observed in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries but not in control or Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries (I–K). Compared to controls and Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries, very few pH3 positive cells were present in precancerous lesions (white dotted lines) of Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries (L–N). Absence of TUNEL staining in pretumoral lesions (white dotted lines) showed that non-proliferating cells in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries are not apoptotic (O–Q). Immunohistochemical analyses showed an increase in p53 accumulation in Amhr2-Cre;Ctnnb1Δ(ex3)/+ tumors compared to controls and loss of p53 expression in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ tumors (R–T). Evidence of DNA damage determined by staining for γ-H2AX and observed in the tumors of both mutants (V & W) but not in the control ovaries (U). The differential expression of p53 was confirmed by Western blot analyses with pooled ovarian lysates (n = 4) and repeated three times (Panel X), which also confirmed the presence of exon 3 deleted β-catenin (arrowhead) in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. Analyses also showed increased expression of pAKT and pS6K, and decreased expression of p21 and p27kip1 in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries. β-actin was used as a loading control. Bars = 50 um.
Mentions: Interactions between Wnt/β-catenin and Akt/PTEN signaling pathways play an important role in carcinogenesis [6]. We examined the status of PTEN by performing immunohistochemical staining for β-catenin and PTEN on serial sections of Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries and observed increased expression of PTEN in pretumoral lesions with nuclear β-catenin (Fig. 3B & E). To examine whether PTEN deletion could affect tumor progression in Amhr2-Cre;Ctnnb1Δ(ex3)/+ ovaries, we developed another mouse model by deleting exon 3 of the β-catenin in PTEN negative cells of the mouse ovary (Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ) (Fig. 3C & F). These mice showed early onset of tumor development and were euthanized because of tumor-related morbidities (Fig. 3G & H). No evidence of ascites or metastases was observed by gross examination of the peritoneal cavity. The ovarian tumors in Amhr2-Cre;Ctnnb1Δ(ex3)/+;PtenΔ/Δ ovaries showed histopathological features similar to the tumors of Amhr2-Cre;Ctnnb1Δ(ex3)/+ mice (Fig. S2). Consistent with previous observations [22], [30], deletion of PTEN alone does not result in ovarian tumor development (data not shown), suggesting that activation of the phosphatidylinositol-3′ kinase (PI3K) signaling pathway with inactivating PTEN mutations alone may not be sufficient for tumor initiation and needs to act in concert with other oncogenes to cause cancer.

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