Superior cancer preventive efficacy of low versus high dose of mTOR inhibitor in a mouse model of prostate cancer

Abstract

The mechanistic target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation and survival in response to the availability of energy sources and growth factors. Cancer development and progression is often associated with constitutive activation of the mTOR pathway, thus justifying mTOR inhibition as a promising approach to cancer treatment and prevention. However, development of previous rapamycin analogues has been complicated by their induction of adverse side effects and variable efficacy. Since mTOR pathway regulation involves multiple feedback mechanisms that may be differentially activated depending on the degree of mTOR inhibition, we investigated whether rapamycin dosing could be adjusted to achieve chemopreventive efficacy without side effects. Thus, we tested the efficacy of two doses of a novel, highly bioavailable nanoformulation of rapamycin, Rapatar, in a mouse prostate cancer model (male mice with prostate epithelium-specific Pten-knockout). We found that the highest efficacy was achieved by the lowest dose of Rapatar used in the study. While both doses tested were equally effective in suppressing proliferation of prostate epithelial cells, higher dose resulted in activation of feedback circuits that reduced the drug's tumor preventive efficacy. These results demonstrate that low doses of highly bioavailable mTOR inhibitor, Rapatar, may provide safe and effective cancer prevention.

Keywords: PTEN; mTOR; prevention; prostate cancer; rapamycin.

Figure 1. Effect of different Rapatar doses on body weight and PCa tumorigenesis in the psPten –/– mouse model.

Groups were treated with vehicle or either 25 or 5 mg/kg Rapatar (Rapa-25 and Rapa-5 groups, respectively) for 8 weeks. Data for individual animals are shown, with the short horizontal black bar indicating the group mean value. (A) Administration of Rapatar does not affect animal growth. Body weights were measured at the end of the treatment period and plotted as a percentage of initial body weight. (B) Low dose Rapatar treatment reduces prostate tumor burden. The ratio of urogenital tract weight (UG) to body weight (BW) at the end of the experiment is plotted. (C) Low dose Rapatar treatment reduces overall urogenital disease. At the end of the experiment, Disease Index values were calculated as total histologic score^*UG weight as described in {Kee, 2004 #123}.

Figure 2. Treatment with low dose Rapatar reduces the number of proliferating cells in the prostate of PCa-prone psPten–/– mice.

Representative anterior prostate lobe (AP), ventral prostate lobe (VP), and lateral prostate lobe (LP) sections stained with anti-Ki67 are shown for (A) Pten-positive mice, (B) psPten–/– mice treated with vehicle, and (C) psPten–/– mice treated with 5 mg/kg Rapatar. Red – Ki67, Blue-DAPI; White arrows indicate Ki67-positive nuclei.

Figure 3. High dose (25 mg/kg) Rapatar treatment promotes development of reactive stroma and induces autophagy.

Markers of reactive stroma (αSMA, panel A) and autophagy (LC3B, panel B) were evaluated by immunostaining of LP sections from untreated Pten+/+ mice and psPten–/– mice treated with vehicle, 25 mg/kg Rapatar or 5 mg/kg Rapatar. In A, red staining indicates αSMA, blue is DAPI co-stain. White arrowheads indicate interruptions in αSMA staining and white asterisks show areas of strong staining for αSMA-positive activated fibroblasts. In B, brown staining indicates LC3B expression; red asterisks highlight areas of strong LC3B expression.

Figure 4. High dose (25 mg/kg) Rapatar treatment of psPten–/– mice leads to increased Akt activation in the liver and the prostate.

Western blot analysis of whole cell extracts (25 ug protein per lane) prepared from the livers (A) and prostates (B) of individual mice treated for 8 weeks with vehicle (n = 3), 25 mg/kg Rapatar (n = 4) or 5 mg/kg Rapatar (n = 4). Membranes were probed with antibodies detecting activated (phosphorylated) and total S6 and Akt proteins. (C) and (D) Quantitation of ratio of activated to total protein for S6 and Akt in livers and prostates respectively based on Western blot signals. Data for individual animals are shown, with the short horizontal black bar indicating the group mean value.

Figure 5. Dysregulation of TGFβ signaling correlates with development of reactive stroma in prostates of psPten –/– mice treated with high dose Rapatar.

Immunostaining of pSMAD2 (panel A, green) and SMA (panel B, red) was performed on LP sections from wild type (WT) mice and psPten–/– mice treated for 8 weeks with vehicle, 25 mg/kg Rapatar or 5 mg/kg Rapatar. Panel (C) shows both markers overlaid with DAPI (blue) staining of nuclei. In A, red and yellow asterisks indicate nuclear stain of pSMAD2 in epithelium and stroma, respectively. In B, blue asterisks indicate areas of smooth muscle layer disintegration and white asterisks indicate appearance of smooth muscle actin in activated stroma. In C, Red arrows point out epithelial cells, yellow arrows point out stromal cells, blue arrows indicate the smooth muscle layer.

Figure 6. Proposed model explaining the differential tumor preventive effects of low and high doses of Rapatar on mTOR signaling in the psPten–/– mouse model.

See details in the text.