An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/- mice

Abstract

PTEN phosphatase acts as a tumor suppressor by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. It is unclear which downstream components of this pathway are necessary for oncogenic transformation. In this report we show that transformed cells of PTEN(+/-) mice have elevated levels of phosphorylated Akt and activated p70/S6 kinase associated with an increase in proliferation. Pharmacological inactivation of mTOR/RAFT/FRAP reduced neoplastic proliferation, tumor size, and p70/S6 kinase activity, but did not affect the status of Akt. These data suggest that p70/S6K and possibly other targets of mTOR contribute significantly to tumor development and that inhibition of these proteins may be therapeutic for cancer patients with deranged PI3K signaling.

Figure 1

Pten^+/− mice develop pheochromocytomas of the adrenal medulla. Morphology of the wild-type adrenal (A) and the Pten^+/− adrenal containing a pheochromocytoma (B). (Magnification, ×40.) The normal medulla can be seen in the center of the wild-type adrenal cortex. Paraffin sections were stained with hematoxylin/eosin. PTEN^+/− animals (mutant) have elevated levels of serum norepinephrine (C) and epinephrine (D) relative to wild type.

Figure 2

Increased proliferation in the neoplastic regions of Pten^+/− uteri and adrenals. Mice were injected with 125 mg/kg of BrdUrd for 1 h before death and sections were stained with an antibody recognizing BrdUrd. (A) Proliferation index in Pten^+/+ (□) and Pten^+/− (■) uteri was calculated by comparing the proliferation index of the secretory epithelium in wild type with that of the CAH. (B) Proliferation index in normal (□) and transformed (■) regions of cysts of Pten^+/− uteri. BrdUrd-positive cells were counted per total number of nuclei. (C) Proliferation index of wild-type (□) and +/− (■) adrenal medulla. Error bars indicate SD. Examples BrdUrd staining of the wild-type (D) and Pten^+/− (E) medulla. Increased BrdUrd incorporation can be seen in E relative to D.

Figure 3

Neoplastic lesions in Pten^+/− uteri have lower levels of Pten, and higher active Akt. (A) Loss of heterozygosity in hyperplastic lesions of the endometrium. Products from wild-type and mutant Pten alleles are amplified in a duplex reaction. Controls consist of products generated from tail DNA isolated from Pten heterozygous (lane 1) and wild-type (lane 2) mice. Lanes 3, 4, and 5 are amplified products from microdissected, hyperplastic endometrial lesions from three Pten heterozygous mice at 32 weeks of age. Loss of the wild-type Pten allele is present in one lesion (lane 3), and both alleles are retained in the other two lesions (lanes 4 and 5). (B) Loss of heterozygosity in Pten^+/− adrenals. Adrenal DNA was prepared from six Pten^+/− mice. After probing the wild-type (wt) and mutant alleles (mut) (arrowheads), we observed that five of the six Pten^+/− adrenals had undergone loss of heterozygosity. Control (+/−) and wild-type DNA (+/+) were prepared from tails. (C–E) Transition zone in Pten^+/−-transformed uterine cysts. Slides were stained with hematoxylin/eosin (C), rabbit polyclonal anti-PTEN (D), and rabbit polyclonal anti-phospho-AKT (Ser-473) (E). (Magnification, ×600.) (F and G) Altered PTEN and phospho-AKT expression are detected in the adrenal medulla. Reduced PTEN staining correlates with transformation and phospho-AKT staining. (F) A small representative focus of reduced PTEN expression in a Pten^+/− adrenal medulla. Notice that most PTEN staining within the medullary cells occurs in the nucleus. (G) A small focus of increased phospho-AKT staining correlates with reduced PTEN expression. (Magnification, ×600.) Cortex (C) stains nonspecifically for PTEN and phospho-AKT.

Figure 4

S6K activity but not AKT phosphorylation can be inhibited with CCI-779. (A) S6K activity in Pten^+/− (●), Pten^+/− treated with CCI-779 for 3 days (○), and Pten^+/+ (□) uterine lysates. Protein concentration was measured from the soluble fraction, and samples were normalized for equal protein concentration before the immunoprecipitation and measurement of S6K activity. (B) Short-term CCI-779 treatment reduces the BrdUrd incorporation index. BrdUrd incorporation index in mock (■)- and drug (□)-treated Pten^+/− uterine epithelium treated for 3 days with vehicle or CCI-779. (C) Phosphorylated S6K levels in 293 cell line and mouse uterine lysates. (Lanes 1 and 2) 293 cells pulsed with epidermal growth factor or starved. Note reduced mobility of S6K in lane 1. Pten^+/+ (lanes 3 and 4) and Pten^+/− (lanes 5–8) uterine lysates analyzed on an 8% polyacrylamide gel. Frozen uteri were ground and transferred into loading SDS buffer, and protein concentrations were normalized by anti-MAPK signal. A slower migrating band was seen in lysates of mice that were not treated with CCI-779 (lanes 7 and 8). This band was not present in Pten^+/− lysates of mice treated with CCI-779 for 3 days (lanes 5 and 6) or in treated or untreated wild-type lysates (lanes 3 and 4). (D) Uterine (Upper) and adrenal (Lower) lysates from wild-type (+/+) and mutant animals (+/−) were resolved on a 4–20% gradient gel, blotted, and probed with anti-phospho-473 and total AKT antibodies. Each sample was collected from a Pten^+/− mouse injected with either diluent or CCI-779 (Drug) for 3 days.

Figure 5

Long-term CCI-779 treatment prevents tumor growth and proliferation in Pten^+/− without affecting Akt activity. (A) Size of neoplastic lesions in mock, untreated (none) and CCI-779-treated (CCI) uteri. All mice are Pten^+/− females and average age of each cohort is indicated. (B) Size of the untreated (none) wild-type (wt), untreated Pten^+/−, mock-treated Pten^+/−, and CCI-779-treated (CCI) Pten^+/− adrenal medullas. (C) Proliferation in mock (■)- and drug (□)-treated uteri. BrdUrd-positive cells were counted per total number of nuclei in CAH. (D) Proliferation in mock-treated Pten^+/− (■) and CCI-779-treated Pten^+/− (□) adrenal medullas. (E and F) Phosoho-473 Akt levels in the mock (E)- and drug (F)-treated uteri. (Magnification, ×400).