The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis - PubMed (original) (raw)

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

K Jiang et al. Mol Cell Biol. 2000 Jan.

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.

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Figures

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/_Hae_III 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/_Hae_III 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.

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The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis - PubMed (original) (raw)