The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis

Abstract

Farnesyltransferase inhibitors (FTIs) represent a novel class of anticancer drugs that exhibit a remarkable ability to inhibit malignant transformation without toxicity to normal cells. However, the mechanism by which FTIs inhibit tumor growth is not well understood. Here, we demonstrate that FTI-277 inhibits phosphatidylinositol 3-OH kinase (PI 3-kinase)/AKT2-mediated growth factor- and adhesion-dependent survival pathways and induces apoptosis in human cancer cells that overexpress AKT2. Furthermore, overexpression of AKT2, but not oncogenic H-Ras, sensitizes NIH 3T3 cells to FTI-277, and a high serum level prevents FTI-277-induced apoptosis in H-Ras- but not AKT2-transformed NIH 3T3 cells. A constitutively active form of AKT2 rescues human cancer cells from FTI-277-induced apoptosis. FTI-277 inhibits insulin-like growth factor 1-induced PI 3-kinase and AKT2 activation and subsequent phosphorylation of the proapoptotic protein BAD. Integrin-dependent activation of AKT2 is also blocked by FTI-277. Thus, a mechanism for FTI inhibition of human tumor growth is by inducing apoptosis through inhibition of PI 3-kinase/AKT2-mediated cell survival and adhesion pathway.

FIG. 1

FTI-277 induces apoptosis in AKT2-overexpressing human cancer cell lines. (A) (Top) Western blot analysis of AKT2 expression in nine ovarian and pancreatic cancer cell lines. Equal amounts of protein were separated by SDS-PAGE and probed with an anti-AKT2 monoclonal antibody. Overexpression of AKT2 was observed in five cell lines (lanes 1 to 5). (Bottom) Western blot analyses of immunoprecipitates prepared from each cell line with monoclonal AKT2 antibody. The blots were detected with polyclonal anti-phospho-Akt-S473 antibody. Elevated levels of phosphorylated AKT2 were detected in AKT2-overexpressing cell lines. (B) Internucleosomal DNA fragmentation. The cells were seeded at 5 × 10⁵ cells/60-mm-diameter dish in 5% FCS medium. After 24 h, the cells were treated with 30 μM FTI-277 (+) or DMSO (−) for 48 h. Genomic DNA was prepared and analyzed on a 1.5% agarose gel as described in Materials and Methods. Lane 1 shows DNA size markers (φX174 replicative-form DNA/HaeIII fragments; GibcoBRL). DNA fragmentation was detected in OVCAR-3, OVCAR-5, OVCAR-8, PANC-1, and ASPC-1 cell lines after FTI-277 treatment.

FIG. 2

Overexpression of wild-type AKT2 sensitizes NIH 3T3 cells to FTI-277-induced apoptosis. A TUNEL assay was used to detect FTI-277-induced apoptosis in AKT2- or H-ras-transformed NIH 3T3 cells in a medium containing 5% FCS (a to c) or 0.1% FCS (d to f). After 48 h of FTI-277 treatment, apoptosis was detected in AKT2-transformed (c) but not H-ras-transformed (b) and pcDNA₃-transfected (a) NIH 3T3 cells in 5% FCS medium. In 0.1% FCS culture medium, FTI-277-induced apoptosis was more prominent in AKT2 (f)- than H-ras (e)-transformed NIH 3T3 cells.

FIG. 3

FTI-277 inhibits AKT2 activation. (A to C) In vitro kinase assay of immunoprecipitates from COS7 cells transfected with HA-AKT2 (A), OVCAR-3 cells (B), and A2780 cells (C). After serum starvation overnight, the cells were treated with or without FTI-277 for 12 h prior to IGF-1 (50 ng/ml) or 5% FCS stimulation for 10 min. (D) FTI-277 does not directly inhibit serum-induced AKT2 activation. An in vitro kinase assay was carried out with immunoprecipitates from OVCAR-3 cells. After serum starvation and restimulation, FTI-277 (30 μM) was directly added into kinase reaction (lane 3). (E) OVCAR-3 cell lysates were analyzed by SDS-PAGE followed by immunoblotting with an anti-lamin B or anti-Rap1A antibody. Lamin B is a substrate for FTase, whereas Rap1A is a substrate for GGTase I. FTI-277 prevented lamin B farnesylation, resulting in a band shift. Rap1A prenylation was unaffected by FTI-277. U, unprenylated form; P, prenylated form (prenylated proteins migrate faster in an SDS-polyacrylamide gel).

FIG. 4

FTI-277 inhibits PI 3-kinase activity. (A and B) In vitro PI 3-kinase assay of the anti-p85 immunoprecipitates from OVCAR-3 and A2780 cells. Following serum starvation overnight, the cells were treated with or without FTI-277 for 12 h prior to IGF-1 (A) or 5% FCS (B) stimulation for 15 min. Basal levels of PI 3-kinase are not significantly different between OVCAR-3 and A2780 cells. However, IGF1- or serum-induced PI 3-kinase activity in both cell lines was inhibited by FTI-277. (C) Western blot analyses of immunoprecipitates from OVCAR-3 cells. Following FTI-277 treatment, the cells were lysed, immunoprecipitated with anti-p85 polyclonal antibody, and detected with an anti-p-Tyr antibody. The blots were reprobed with anti-p85 antibody. (D) FTI-277 does not directly inhibit serum-induced PI 3-kinase activation. An in vitro PI 3-kinase assay was carried out with immunoprecipitates from OVCAR-3 cells. After serum starvation and restimulation, FTI-277 (30 μM) was directly added to the kinase reaction (lane 3).

FIG. 5

AKT2 phosphorylates recombinant BAD and BADS112A but not BADS136A or BAD2SA: inhibition by FTI-277 in vitro and in vivo. (A) In vitro kinase assays using anti-HA immunoprecipitates from COS7 cells transfected transiently with HA-tagged AKT2 constructs expressing wild-type AKT2 (HA-AKT2), constitutively active AKT2 (Myr-AKT2), and dominant-negative mutant AKT2 (AKT2-E299K). Following serum starvation, the cells were treated with or without FTI-277 (3 h) or wortmannin (30 min) prior to IGF-1 (50 ng/ml) stimulation. Anti-HA immunoprecipitates were subjected to an in vitro kinase assay using wild-type BAD (WT-Bad), BADS112A, BADS136A, or BAD2SA as the substrate. Note that wortmannin inhibited BAD phosphorylation more efficiently than FTI-277 due to relatively short time of treatment of the cells with FTI-277 (3 h; see Fig. 8). (B) Western blot analysis of phosphorylation (top) and expression (bottom) of endogenous BAD from parental OVCAR-3 cells and the stably transfected cell clones expressing Myr-AKT2 treated with or without FTI-277 before stimulation with IGF-1 (50 ng/ml) for 10 min. (C) Western blot analyses of cell lysates from ovarian and pancreatic cancer cell lines. The blots were detected by anti-BAD (top) or anti-p-BAD (bottom) antibody.

FIG. 6

Activation of AKT2 following attachment to fibronectin: inhibition by FTI-277. OVCAR-3 cells were serum starved, treated with or without FTI-277 or LY294002, and replated on fibronectin- or polylysine-coated plates. AKT2 was immunoprecipitated with an anti-AKT2 monoclonal antibody, and the immunoprecipitates were subjected to an in vitro kinase assay using histone H₂B as the substrate. The autoradiogram (top) and quantitation by phosphorimaging (bottom) show that AKT2 is activated by cell adhesion to fibronectin (lane 2) but not to polylysine (lane 1). Integrin-mediated AKT2 activation is inhibited by LY294002 (lane 3) and FTI-277 (lane 4).

FIG. 7

A constitutively activated form of AKT2, but not treatment with IGF-1, rescues human cancer cells from FTI-277-induced apoptosis. (A) OVCAR-3 cells were transfected with pcDNA₃ or constitutively active AKT2 (Myr-HA-AKT2), and two stable clones were established. Western blot analyses with an anti-HA antibody revealed expression of Myr-AKT2 in these two clonal cell lines but not in cells transfected with pcDNA₃ vector alone. (B and C) DNA fragmentation and TUNEL assay. After treatment with FTI-277, the DNA ladder and apoptotic cells were not observed in Myr-AKT2-transfected cells (lane 4 of panel B and middle column of panel C). However, FTI-277-induced apoptosis was detected in pcDNA₃-transfected OVCAR-3 cells cultured in medium containing either IGF-1 (50 ng/ml; lane 5 of panel B and rightmost column of panel C) or 5% FCS (lane 3 of panel B and leftmost column of panel C). Lane 1, φX174 replicative-form DNA/HaeIII markers; lanes 2 and 3, parental OVCAR-3 cells treated with vehicle (DMSO) and FTI-277, respectively.

FIG. 8

FTI-277 targets a short-lived farnesylated protein, but not RhoB, upon inhibition of AKT2 activation. (A) In vitro kinase assay of the AKT2 immunoprecipitates from OVCAR-3 cells. The cells were treated with FTI-277 at different time points as indicated at the top, serum starved, stimulated with IGF-1, lysed, and immunoprecipitated with monoclonal anti-AKT2 antibody. The immunoprecipitates were subjected to in vitro kinase assay (top), and the filter was detected with polyclonal anti-AKT2 antibody (bottom). (B) In vitro kinase assays of HA-AKT2 immunoprecipitated from lysates of COS7 cells transfected with HA-AKT2 and different combinations of v-Ha-Ras or wild-type (WT) and active (V14) forms of RhoB.