An inducible knockout mouse to model the cell-autonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias

Abstract

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ER(T) under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors.

Fig. 1.

Differential Cre-ER^T activity and PTEN deletion in endometrial epithelial and stromal compartments. (A) Schematic showing the experimental setup for tamoxifen (TAM) treatment. Briefly, mice were weaned 3 weeks after birth and genotyped. At 4–5 weeks old, animals were given a single intraperitoneal dose of tamoxifen, or corn oil as a vehicle (con). Mice were sacrificed 7 days later. (B) Tamoxifen induces a dose-dependent deletion of PTEN. PTEN immunohistochemistry corresponding to CAG-Cre-ER^T+/−PTEN^fl/fl 7 days after tamoxifen injection. Note that stromal cells remained positive for PTEN staining. (C) Quantification of the percentage of endometrial epithelial cells displaying negative for PTEN immunostaining after injection of the indicated doses of tamoxifen. (D) Analysis of tamoxifen-induced recombination in CAG-Cre-ER^T mT/mG reporter mice. Representative images corresponding to CAG-Cre-ER^T+/−mT/mG^+/− uterus 1 week after tamoxifen injection are shown. Note that, upon tamoxifen treatment, only epithelial cells showed a switch from red to green fluorescence.

Fig. 2.

Tamoxifen-induced PTEN deletion causes rapid and reproducible development of endometrial hyperplasia and in situ adenocarcinoma of the endometrium. (A–D) Comparison of histopathology and PTEN expression of Cre-ER^T+/−PTEN^fl/fl mice injected with tamoxifen (TAM) or corn oil (CON) at different time points. (A–C) Representative images showing PTEN immunohistochemistry (bottom panels) and H&E staining (top panels) of uterus from mice sacrificed in weeks 0–2 (A), weeks 3–5 (B) or weeks 6–8 (C) after tamoxifen injection. (D) Graph showing the percentages of endometrial lesions distributed by periods of time.

Fig. 3.

Evaluation of inducible and tissue-specific Cre-ER^T recombinase activity in the double-fluorescent mT/mG reporter mice. CAG-Cre-ER^−/−mT/mG^+/− and CAG-Cre-ER^T+/− mT/mG^+/− mice were given a single dose of tamoxifen as described in Fig. 1. After 7 days, different tissues were fixed and analyzed for the presence of green (indicative of Cre-ER recombination) or red (indicative of lack of Cre-ER recombination) fluorescence. (A) Representative confocal images corresponding to organs containing epithelial tissues (thyroid, liver, colon, kidney and lung) showing green fluorescence after tamoxifen injection. (B) Representative images of hematopoietic and lymphoid organs (bone marrow and spleen). Note absence of green fluorescence.

Fig. 4.

Tamoxifen-induced PTEN deletion causes the rapid development of prostate intraepithelial neoplasia (PIN). Prostates from CAG-Cre-ER^T−/−PTEN^fl/fl or CAG-Cre-ER^T+/−PTEN^fl/fl were analyzed 6–8 weeks after tamoxifen (TAM) or corn oil (CON) injection. (A) Representative images of CAG-Cre-ER^T+/−PTEN^fl/fl tamoxifen-treated males showing high-grade PIN lesions and PTEN-negative immunostaining. Control mice displayed normal histology and retained PTEN immunostaining. (B) Representative images of cytokeratin staining, showing increased immunostaining in high-grade PIN epithelial cells. (C) Quantification of the percentage of mice displaying prostatic lesions 6–8 weeks after tamoxifen injection. (D) Representative images (top) and quantification (bottom) of Ki-67 immunostaining in prostates of CAG-Cre-ER^T–/–PTEN^fl/fl mice injected with tamoxifen, and CAG-Cre-ER^T+/−PTEN^fl/fl mice injected with corn oil (CON) or tamoxifen (TAM). **P≤0.001.

Fig. 5.

PTEN inactivation leads to the development of thyroid hyperplasia. (A) Comparison of representative thyroids from CAG-Cre-ER^T+/−PTEN^fl/fl mice 6–8 weeks after being injected with corn oil (CON) or tamoxifen (TAM). (B) Representative images of hyperplasia in thyroids dissected from three corn-oil-injected (CON) and three tamoxifen-injected (TAM) mice. (C) Immunofluorescence staining showing PTEN loss in CAG-Cre-ER^T+/−PTEN^fl/fl thyroids. Representative images showing PTEN immunofluorescence of mice injected with corn oil displaying no hyperplasic nodules (CON), mice injected with corn oil displaying hyperplasic nodules (CON-Nodule) or mice injected with tamoxifen (TAM). Arrow indicates PTEN-negative immunofluorescence in hyperplasic module.

Fig. 6.

PTEN loss does not cause rapid neoplastic growth in other epithelia or in hematopoietic or lymphoid tissues. Representative images of H&E staining and PTEN immunohistochemistry corresponding to (A) kidney, (B) liver, (C) lung, (D) colon, (E) bone marrow and (F) spleen from CAG-Cre-ER^T+/−PTEN^fl/fl mice 8 weeks after injection with corn oil (CON) or tamoxifen (TAM).

Fig. 7.

mTOR inhibition reduces endometrial lesions and thyroid hyperplasia. (A) Schematic representation of the protocol used for everolimus administration. Briefly, 5 weeks after tamoxifen injection, animals started to receive a single daily intraperitoneal dose of everolimus for 14 consecutive days and then they were sacrificed. (B) At 7 weeks after tamoxifen injection (TAM), mice exhibited lethargy, ruffled fur and hunched posture. Animals treated with everolimus (TAM+EVE) remained healthy and active, similar to controls (CON). (C) H&E staining and PTEN immunohistochemistry images corresponding to endometria from mice treated with corn oil (CON), tamoxifen (TAM) or tamoxifen plus everolimus (TAM+EVE). Everolimus-treated females showed reduced lesions and PTEN-negative healthy areas. Percentage of animals developing endometrial hyperplasia (EH) and endometrial in situ adenocarcinomas (EC) after injection of corn oil (CON), tamoxifen (TAM) or tamoxifen and everolimus (TAM+EVE) are shown in the top graph. Representative images (bottom left) and quantification (bottom right) of Ki-67 immunostaining performed on uterus dissected from mice injected with corn oil (CON), tamoxifen (TAM) or tamoxifen plus everolimus (TAM+EVE). (D) Representative images of thyroids dissected from mice injected with corn oil (CON), tamoxifen (TAM) or tamoxifen plus everolimus (TAM+EVE). Everolimus treatment also reduced thyromegaly in tamoxifen-injected mice. Everolimus caused a marked decrease of the index of proliferation, with levels similar to control animals, as assessed by Ki-67 immunostaining. *P≤0.05; **P≤0.001.