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. 2001 Aug 28;98(18):10314-9.
doi: 10.1073/pnas.171076798. Epub 2001 Aug 14.

Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR

M S Neshat  1 , I K Mellinghoff, C Tran, B Stiles, G Thomas, R Petersen, P Frost, J J Gibbons, H Wu, C L Sawyers
Affiliations

Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR

M S Neshat et al. Proc Natl Acad Sci U S A. .

Abstract

Recent evidence places the FRAP/mTOR kinase downstream of the phosphatidyl inositol 3-kinase/Akt-signaling pathway, which is up-regulated in multiple cancers because of loss of the PTEN tumor suppressor gene. We performed biological and biochemical studies to determine whether PTEN-deficient cancer cells are sensitive to pharmacologic inhibition of FRAP/mTOR by using the rapamycin derivative CCI-779. In vitro and in vivo studies of isogenic PTEN(+/+) and PTEN(-/-) mouse cells as well as human cancer cells with defined PTEN status showed that the growth of PTEN null cells was blocked preferentially by pharmacologic FRAP/mTOR inhibition. Enhanced tumor growth caused by constitutive activation of Akt in PTEN(+/+) cells also was reversed by CCI-779 treatment, indicating that FRAP/mTOR functions downstream of Akt in tumorigenesis. Loss of PTEN correlated with increased S6 kinase activity and phosphorylation of ribosomal S6 protein, providing evidence for activation of the FRAP/mTOR pathway in these cells. Differential sensitivity to CCI-779 was not explained by differences in biochemical blockade of the FRAP/mTOR pathway, because S6 phosphorylation was inhibited in sensitive and resistant cell lines. These results provide rationale for testing FRAP/mTOR inhibitors in PTEN null human cancers.

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Figures

Figure 1
Figure 1
PTEN null cells have enhanced sensitivity to mTOR inhibition. Isogenic MEFs derived from PTEN knockout mice were cultured for 72 h in the presence of vehicle alone (open bars), 1.0 nM CCI-779 (hatched bars), or 10 nM CCI-779 (solid bars). Cell growth was measured by cell counts determined by Trypan blue staining. The data are plotted as the number of cells relative to vehicle control for three experiments. The IC50 for growth inhibition of the cell lines in the table was determined by 3H-uptake studies. (*IC50 for 9L was obtained based on lack of growth inhibition by up to 10 nM of CCI-779 as measured by direct cell count.) Examples are shown for U87MG and MDA-435. The PTEN status of these lines has been reported previously (26, 35) with the exception of SF-268, SF-295, and SF-539, which were kindly provided by R. Parsons (personal communication).
Figure 2
Figure 2
Enhanced S6 kinase activation in PTEN null tumor lines. (A) Phosphorylated (Ser-235/236) and total S6 protein and actin were measured by immunoblot in 9L and U87MG cells treated with vehicle or doses of 0.1, 1.0, and 10 nM CCI-779 for 7 h. Serum challenge was for 15 min. Equal amounts of protein were loaded per lane as determined by Bio-Rad DC protein assay. (B) S6 kinase activity was measured by in vitro kinase assay (31) in MDCK cells stably transfected with vector or wild-type PTEN (Left) and in LAPC-4 prostate cancer cells stably infected with retrovirus expressing myr-Akt or vector control (Right). MDCK cells were serum-starved overnight, pretreated with vehicle (open bars) or 10 nM of CCI-779 (solid bars), and then challenged with serum for 15 min. LAPC-4 cells were treated identically except that no serum challenge was given. Results from two experiments are plotted relative to serum-starved, untreated cells. Expression of PTEN and Akt in the transfectants was confirmed by immunoblot (data not shown). (C) Levels of total and activated Akt and MAPK were measured by immunoblot in lysates of PTEN wild-type (DU145) and PTEN null (PC3) cells by using specified antibodies after 2 h of pretreatment with vehicle or 10 nM CCI-779.
Figure 3
Figure 3
PTEN null cells have enhanced sensitivity to mTOR inhibition in vivo. (A) PTEN+/+ or PTEN−/− ES cells were injected s.c. into nude mice at a dose of 5 × 105 cells per mouse (n = 20). When tumor volume reached 200 mm3, mice were randomized to treatment with vehicle or 40 mg/kg CCI-779. The fold change in tumor volume in response to treatment was plotted. (B) Single-cell suspensions of LAPC-4 or LAPC-9 prostate cancer xenografts were injected s.c. into male SCID mice (n = 80) at a dose of 106 cells per mouse. When tumors became palpable, mice were randomized (arrow) to treatment with vehicle, 0.1 mg/kg, 4 mg/kg, or 40 mg/kg CCI-779. The fold change in tumor volume from two independent experiments is plotted. (C) Tumors were harvested from mice after 5 days of treatment with vehicle or 0.1 mg/kg CCI-779 and lysed by boiling in 2% SDS buffer. Immunoblots were performed by using antibodies for phosphorylated S6 (Ser-235/236), total S6, and actin.
Figure 4
Figure 4
Effect of CCI-779 on cell size, proliferation, and apoptosis. Photomicrographs (×200) of hematoxylin/eosin- and Ki-67-stained sections of LAPC-4 and LAPC-9 tumors are shown after 5 days of treatment with vehicle or CCI-779. Cell size was measured by morphometry (1,200 total cells on 20 sections). Proliferation and apoptosis were measured by counting the number of Ki-67-positive nuclei or apoptotic bodies [terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive] divided by high-power field (hpf) (12–18 hpf/tumor, 5 tumors/group). All values are mean ± SEM. Both cell size and proliferation were reduced significantly in CCI-779-treated LAPC-9 tumors but not LAPC-4 tumors (LAPC-4 size: P = 0.95; LAPC-9 size: P = 0.001; LAPC-4 proliferation: P = 0.212; LAPC-9 proliferation: P = 0.01, Student's t test). Apoptosis was increased significantly in treated LAPC-4 and LAPC-9 tumors (P = 0.01 and P = 0.005, respectively, Student's t test). The relative effect of CCI-779 treatment on size, proliferation, and apoptosis in LAPC-4 vs. LAPC-9 was compared by using an ANOVA model, with P values shown above the bar graphs.
Figure 5
Figure 5
Akt-mediated growth in PTEN wild-type tumor cells is mTOR-dependent. LAPC-4/puro or LAPC-4/myr-Akt cells were injected s.c. into male SCID mice (n = 20) at a dose of 106 cells per mouse. When tumor volume reached 50–200 mm3, mice were randomized to treatment with vehicle or 0.1 mg/kg CCI-779, given by i.p. injection for 5 consecutive days in weeks 1 and 4. The fold changes in tumor volume (Left) and in serum prostate-specific antigen (PSA) (Right) are plotted.
Figure 6
Figure 6
Analysis of S6, 4E-BP1, and cyclin D1 in PTEN+/+ and PTEN−/− MEFs. PTEN+/+ or PTEN−/− MEFs were treated with the indicated concentrations of CCI-779 for 6 h, lysed by three freeze/thaw cycles in 50 mM Tris, pH 7.5/150 mM KCl mM EDTA/1 mM EGTA/1 mM DTT/50 mM 2-mercaptoethanol, supplemented with protease and phosphatase inhibitors, and probed with antibodies to phosphorylated S6, total S6, total 4E-BP1, or actin. The level of 4E-BP1 bound to eIF4E was measured by precipitation of eIF4E by using 7methyl-GTP Sepharose, followed by 4E-BP1 immunoblot. Comparable precipitation of eIF4E was confirmed by immunoblot.
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