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. 2002 Jun;22(11):3842-51.
doi: 10.1128/MCB.22.11.3842-3851.2002.

Essential role of AKT-1/protein kinase B alpha in PTEN-controlled tumorigenesis

Bangyan Stiles  1 , Valeriya Gilman, Natalya Khanzenzon, Ralf Lesche, Annie Li, Rong Qiao, Xin Liu, Hong Wu
Affiliations

Essential role of AKT-1/protein kinase B alpha in PTEN-controlled tumorigenesis

Bangyan Stiles et al. Mol Cell Biol. 2002 Jun.

Abstract

PTEN is mutated at high frequency in many primary human cancers and several familial cancer predisposition disorders. Activation of AKT is a common event in tumors in which the PTEN gene has been inactivated. We previously showed that deletion of the murine Pten gene in embryonic stem (ES) cells led to increased phosphatidylinositol triphosphate (PIP(3)) accumulation, enhanced entry into S phase, and better cell survival. Since PIP(3) controls multiple signaling molecules, it was not clear to what degree the observed phenotypes were due to deregulated AKT activity. In this study, we mutated Akt-1 in Pten(-/-) ES cells to directly assess the role of AKT-1 in PTEN-controlled cellular processes, such as cell proliferation, cell survival, and tumorigenesis in nude mice. We showed that AKT-1 is one of the major downstream effectors of PTEN in ES cells and that activation of AKT-1 is required for both the cell survival and cell proliferation phenotypes observed in Pten(-/-) ES cells. Deletion of Akt-1 partially reverses the aggressive growth of Pten(-/-) ES cells in vivo, suggesting that AKT-1 plays an essential role in PTEN-controlled tumorigenesis.

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Figures

FIG.1.
FIG.1.
Inactivation of mouse Akt-1 gene. (A) A restriction map of the mouse Akt-1 locus is shown in the top panel with exons depicted. The middle panel shows the targeting vector with exons 2 and 3 deleted and replaced with a PGKpuro cassette. The bottom panel is the predicted recombinant harboring the deletions with the position of the 3′ external probe indicated below it. (B) Southern blot analysis of the Pten and Akt-1 locus. (C) Western analysis of PTEN and AKT-1 levels in the indicated ES cell clones. Cell lysates (20 μg) were run on a polyacrylamide gel and Western blotted with antibodies for PTEN (top) and AKT-1 (bottom). (D) Western blot analyses of phospho-AKT, ERK, GSK-3α/β, and p70S6 kinase status in indicated ES clones. Cell lysates (20 μg) were run on a polyacrylamide gel and Western blotted with antibodies for the different molecules. The same blots were reblotted with vinculin (Vinc) or actin as loading controls. (E) Serum-starved ES cells were treated with either 1 μg of IGF-1/ml or 15% fetal calf serum for 10 or 30 min. Cell lysates were analyzed for phospho-AKT (upper panels) and GSK-3 (lower panels) status. The same blots were also reblotted with actin as loading controls (data not shown). pGSK3a/b, pGSK3α/β.
FIG. 2.
FIG. 2.
Cell survival potential and apoptotic signals in ES clones carrying Pten/Akt deletions. (A) Survival of ES cells in response to serum starvation. ES cells (5 × 104) were seeded the day before serum withdrawal. Cells were cultured without serum for 4 days. Cell survival was observed under a light microscope and quantified by counting the number of surviving cells under each culture condition. Photographs are representative of three independent experiments. Left panels, cells grown under serum-free condition. Right panels, cells grown under normal growth conditions (15% serum). (B) Quantification of cell numbers at different serum concentrations. Data presented are means ± standard errors of the means of n = 3. Bars designated with the letter “a” are statistically significantly different from the WT bars at the same serum concentration (P ≤ 0.05). Bars designated with the letter “b” are statistically significantly different from the Pten−/− bars at the same serum concentration (P ≤ 0.05). (C) Quantification of cell numbers with or without caspase inhibitor cocktail. Data are presented as fold increases over cell numbers in the WT cultures in the absence of the inhibitor cocktail. (D) Western blot analyses of the phosphorylation status of FKHRL1, FKHR, and BAD under normal culture conditions. Total cell lysates (20 μg) were run on a polyacrylamide gel and Western blotted with antibodies for phosphor-FKHR/FKHRL1 and BAD. The same blots were also blotted with vinculin (vinc) as controls. (E) Serum-starved ES cells were treated with either 1 μg of IGF-1/ml or 15% fetal calf serum for 10 or 30 min. Cell lysates were then analyzed for phospho-FKHR status by Western blotting (upper panels). The same blots were also blotted with actin as controls (lower panels).
FIG. 3.
FIG. 3.
Growth potential, cell cycle profile, and analysis of cell cycle regulators in Pten/Akt clones. (A) Growth curve of indicated ES clones. (B) Growth competition assay. Pten−/−;Akt−/− ES cells were cocultivated with equal numbers of WT or Pten−/− cells. DNA was extracted from the cocultures after each passage and was analyzed with Southern analysis to determine the ratio of WT Akt allele (23 kb) to mutant Akt allele (15 kb). Lanes 1 and 2, WT and Pten−/−;Akt−/− cells cultured alone. Lanes 3 to 6, Pten−/−;Akt−/− ES cells cocultured with WT cells at different passages (p1 to p4). Lanes 7 to 10, Pten−/−;Akt−/− ES cells cocultured with Pten−/− cells at different passages (p1 to p4). KO, knockout. (C) Western blot analysis of cell cycle regulators. Total cell lysates (20 μg) were separated on sodium dodecyl sulfate-polyacrylamide gels and blotted with cell cycle inhibitors p27KIP1 and p16INK1 as well as cyclins A, D1, and E. Vinculin (vinc) was used on each blot as a loading control. (D) Cell cycle analysis. ES cells were synchronized at M phase with Colcemid treatment followed by replating in Colcemid-free medium to allow reentry into the cell cycle. FACS analysis was performed on each clone at different time points (2 and 3 h), and the percentages of cells remaining in M phase are shown here.
FIG. 4.
FIG. 4.
Deleting Akt-1 in addition to Pten delays the onset of teratoma formation and decreases the sizes of tumors formed by Pten−/− ES cells. WT, Pten−/−;Akt+/+, and Pten−/−;Akt−/− cells (5 × 106) were injected subcutaneously onto the backs of immunoincompetent nude mice. Each mouse was injected bilaterally with cells carrying a different genotype. Teratoma formations were observed and recorded at indicated time points. (A) Examples of teratomas growing on the backs of nude mice. (B) Onset of teratomas formed from different ES clones. Each dot represents one animal; n = 9 in each group. The average time for tumor appearance is indicated as a line with the actual number beside it. (C) Progression of teratomas formed by different ES clones. The day of tumor detection is set as day 0. Data are presented as means ± standard errors of the means of n = 5. (D) Histology analysis of teratomas harvested from the nude mice. Left panel, tumors formed by injecting WT ES cells showed well-differentiated tissues with skin characteristics (arrow); middle panel, teratomas formed by Pten−/− ES cells are highly proliferative, less differentiated, and well vascularized (arrow); right panel, tumors formed by injection of Pten−/−;Akt−/− ES cells. Deletion of Akt-1 in addition to Pten partially reverses the aggressive growth of Pten−/− teratomas. The arrow indicates the more differentiated keratinized skin, similar to tumors generated by WT ES cells (left panel).
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