Geranylgeranyltransferase I inhibitors target RalB to inhibit anchorage-dependent growth and induce apoptosis and RalA to inhibit anchorage-independent growth

Abstract

Geranylgeranyltransferase I inhibitors (GGTIs) are presently undergoing advanced preclinical studies and have been shown to disrupt oncogenic and tumor survival pathways, to inhibit anchorage-dependent and -independent growth, and to induce apoptosis. However, the geranylgeranylated proteins that are targeted by GGTIs to induce these effects are not known. Here we provide evidence that the Ras-like small GTPases RalA and RalB are exclusively geranylgeranylated and that inhibition of their geranylgeranylation mediates, at least in part, the effects of GGTIs on anchorage-dependent and -independent growth and tumor apoptosis. To this end, we have created the corresponding carboxyl-terminal mutants that are exclusively farnesylated and verified that they retain the subcellular localization and signaling activities of the wild-type geranylgeranylated proteins and that Ral GTPases do not undergo alternative prenylation in response to GGTI treatment. By expressing farnesylated, GGTI-resistant RalA and RalB in Cos7 cells and human pancreatic MiaPaCa2 cancer cells followed by GGTI-2417 treatment, we demonstrated that farnesylated RalB, but not RalA, confers resistance to the proapoptotic and anti-anchorage-dependent growth effects of GGTI-2417. Conversely, farnesylated RalA but not RalB expression renders MiaPaCa2 cells less sensitive to inhibition of anchorage-independent growth. Furthermore, farnesylated RalB, but not RalA, inhibits the ability of GGTI-2417 to suppress survivin and induce p27(Kip1) protein levels. We conclude that RalA and RalB are important, functionally distinct targets for GGTI-mediated tumor apoptosis and growth inhibition.

FIG. 1.

RalA-CCIL and RalB-CCLL are geranylgeranylated, whereas the mutants RalA-CCIS and RalB-CCLS are farnesylated. (A) Ral DNAs were used in transcription-translation-prenylation assays using either radiolabeled [³⁵S]methionine, [³H]FPP, or [³H]GGPP and then run on an SDS-PAGE and visualized by autoradiography as described in Materials and Methods. Results are representative of two independent experiments. (B) RalA-CCIS, RalA-CCIL, RalB-CCLS, and RalB-CCLL DNAs were transcribed, translated, and prenylated using either [³⁵S]methionine, [³H]FPP, or [³H]GGPP in the presence of either vehicle (DMSO), 250 nM FTI-2148, or 250 nM GGTI-2418 and then run on an SDS-PAGE and visualized by autoradiography. Results are representative of two independent experiments.

FIG. 2.

Geranylgeranylated (GG) and farnesylated (F) RalA and RalB localize similarly and require prenylation for correct localization. Cos7 cells grown on coverslips were transiently transfected with plasmids expressing the indicated proteins and treated with the indicated inhibitors (25 μΜ) and then analyzed for FLAG-Ral or HA-H-Ras distribution using immunoflorescence as described in Materials and Methods. Nuclei were visualized using DAPI stain. Results are representative of three independent experiments.

FIG. 3.

Farnesylated and geranylgeranylated RalA and RalB are equivalent in mediating activation of NF-κB promoter activity. (A) Cos7 cells were transiently transfected with the indicated plasmids, and membrane (M) and cytosolic (C) fractions were isolated and probed by Western blotting. HSP90 was used as a cytosolic fraction marker. Results are representative of two independent experiments. (B) NIH 3T3 cells were serum starved and transiently cotransfected with plasmid expressing the indicated proteins and an NF-κB reporter plasmid. NF-κB promoter activity was detected following transfection as described in Materials and Methods. Expression was analyzed by Western blot analysis. Results are representative of three independent experiments.

FIG. 4.

Ectopic expression of farnesylated RalB, but not RalA, renders cells less sensitive to GGTI-2417 inhibition of survival and proliferation and induction of apoptosis in Cos7 cells. (A and B) Inhibition of RalB prenylation is required for GGTI induction of cell death and inhibition of proliferation. Cos7 cells were transfected with the indicated plasmids and then treated with GGTI-2417 or vehicle (DMSO) control. Cell viability was determined by the trypan blue dye exclusion assay. Data shown are the averages of three independent experiments. (C) Cell lysates from panels A and B were analyzed for expression by Western blot analysis as described in Materials and Methods. (D) Inhibition of RalB prenylation is required for GGTI induction of apoptosis. Cos7 cells were transiently transfected with plasmids expressing the indicated proteins and then treated for 72 h with 50 μM GGTI-2417 or DMSO control, collected, and analyzed for apoptosis via a TUNEL assay as described in Materials and Methods. Data shown are the averages of three independent experiments. *, P < 0.05.

FIG. 5.

Stable expression of farnesylated RalB, but not RalA, promotes resistance to the antiproliferative and proapoptotic effects of GGTI-2417 in MiaPaCa2 cells. (A) Dose-response results to determine the efficacy of GGTI-2417 in inhibiting RalA and RalB prenylation in MiaPaCa2 cells. MiaPaCa2 cells were treated with various concentrations of GGTI-2417 for 48 h and processed for Western blotting as described in Materials and Methods. (B) Stable MiaPaCa2 cell lines were created using retroviral infection of a puromycin resistance marker along with the indicated transgenes as described in Materials and Methods. Cells were lysed, and expression was assessed by Western blot analysis. (C) Stably expressing MiaPaCa2 cells were treated with the indicated concentrations of GGTI-2417 for 96 h. Proliferation was measured by the MTT viability assay as described in Materials and Methods. Data shown are the averages of at least three independent experiments. (D) MiaPaCa2 cells stably expressing the indicated Ral proteins were treated with the indicated concentrations of GGTI-2417 for 72 h. Proliferation was assessed by trypan blue dye exclusion assay as described in Materials and Methods. Data shown are the averages of at least three independent experiments. (E) MiaPaCa2 cells stably expressing the indicated Ral proteins were treated with GGTI-2417 (30 μΜ) and lysed, and expression was assessed by Western blot analysis. Data shown are representative of three independent experiments. (F) MiaPaCa2 cells stably expressing the indicated Ral proteins were treated with GGTI-2417 for 48 h, and apoptosis was assessed by a TUNEL assay as described in Materials and Methods. Data shown are the averages of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 6.

Stable expression of farnesylated RalA, but not RalB, induces resistance to inhibition of anchorage-independent growth by GGTI-2417 in MiaPaCa2 cells. MiaPaCa2 cells stably expressing the indicated Ral proteins were seeded into 12-well plates in 0.3% soft agar and treated with the indicated concentrations of GGTI-2417 for 10 days as described in Materials and Methods. Data shown are the averages of two independent experiments repeated in triplicate. *, P < 0.05; ***, P < 0.001.

FIG. 7.

RalB-F, but not RalA-F, inhibits the ability of GGTI-2417 to increase p27^Kip1 and decrease survivin protein levels. Stably expressing MiaPaCa2 cells were seeded into six-well plates and treated with GGTI-2417 (30 μΜ) for 48 h. Cells were lysed, and expression was assessed by Western blot analysis. Data shown are representative of three independent experiments.