GGTI-2417 induces a concentration-dependent increase of p27 protein levels, G₀/G₁ phase accumulation, inhibition of proliferation, and cell death in breast cancer cells. MDA-MB-468 breast cancer cells were treated with compound or vehicle control for 48 h and then processed for various assays. (A) Effect of increasing concentrations of GGTI-2417 or 25 μM FTI-2153 on unprenylated Rap1 (U-Rap1), HDJ-2, and p27 levels. The U-Rap1 and p27 bands were quantified by densitometry. The results are given above the images as percent unprenylated Rap1 (% U) compared to the maximum seen at 100 μM GGTI-2417 or as fold change compared to the control in lane 1, respectively. (B) Effect of increasing concentrations of GGTI-2417 on the distribution of RhoA between plasma membrane and cytosolic fractions. (C) Changes in p27 levels in response to GGTI-2417 over time. The p27 bands were quantified by densitometry, and the change is indicated above the image. (D) Trypan blue exclusion assays to determine cell proliferation and cell death. Standard deviations from three independent experiments are shown with error bars. (E) Effect of increasing concentrations of GGTI-2417 on PARP cleavage. (F) MDA-MB-468, MDA-MB-231, SK-Br3, and BT-474 breast cancer cells were treated with 50 μM of GGTI-2417 or vehicle and then tested by Western blot assays for unprenylated Rap1, HDJ-2, and p27. U and P designate unprocessed and processed forms of the prenylated proteins, respectively.

GGTI-2417 inhibits phosphorylation events that are required for subsequent p27 degradation and accumulates nuclear p27. (A) MDA-MB-468 cells were treated with 50 μM GGTI-2417 and processed as described in the legends for Fig. 2 and 3. Western blot analysis of immunoprecipitated p27 with a p27 or phosphotyrosine antibody, followed by densitometric analysis, revealed that p27 levels increased 11.9-fold, while Tyr phosphorylation increased by only 3.5-fold, suggesting a specific downregulation of Tyr74 and/or Tyr88 phosphorylation (top panel). There are three Tyr residues in p27, in positions 74, 88, and 89, but only Tyr74 and Tyr88 are phosphorylated (12, 23). GGTI-2417 caused an even greater downregulation of Thr187 phosphorylation in p27. (B) Inhibition of protein synthesis with cycloheximide (10 μg/ml) 2 h prior to, and during, exposure to 50 μM GGTI-2417 does not prevent the increase in p27 levels. The changes compared to the control, as determined by densitometry, are given above the image. (C) Representative immunofluorescence images showing that a 48-h exposure of MDA-MB-468 cells to GGTI-2417 upregulates nuclear p27, where it can function as a Cdk inhibitor. (D) Quantitative analysis of the relative cellular amounts of p27 in the nucleus and cytoplasm. Five hundred ninety-five vehicle-treated cells in six fields and 447 GGTI-2417-treated cells in 11 fields were analyzed. The plotted values were computed with the following formula: number of pixels × intensity/number of cells. The value for vehicle-treated cells was set at 100%. The columns represent the mean ± standard error of the mean. +, present; − absent.

GGTI-2418 and its prodrug GGTI-2417 are potent and selective inhibitors of GGTase I activity in vitro and in whole cells. (A) Structures of GGTI-2417/GGTI-2418, GGTI-2431/GGTI-2432, and GGTI-2429/GGTI-2430. (B) Increasing concentrations of GGTI-2418, GGTI-2432, and GGTI-2430 were incubated in the presence of H-Ras-CVLL protein and [³H]geranylgeranylpyrophosphate to determine inhibition of GGTase I activity (•) and H-Ras-CVLS protein and [³H]farnesylpyrophosphate to determine inhibition of FTase activity (○), as described in Materials and Methods. (C) In vitro GGTase I competition assay showing the activity against H-Ras-CVLL alone (⋄) or in the presence of 10 nM GGTI-2418 (•) and 20 nM GGTI-2418 (▴). (D) H-Ras-transformed NIH-3T3 cells were treated in the absence or presence of GGTI compounds for 48 h and processed by Western blotting with antibodies to Rap1 and H-Ras. U indicates the band for unprenylated H-Ras or Rap1 protein, and P indicates the band for fully prenylated H-Ras or Rap1 protein.

p27 is required for the induction of cell death by GGTI-2417. (A to C) siRNA-mediated silencing of p27. MDA-MB-468 cells were treated with 50 μM GGTI-2417, 1 μM taxol, or vehicle (DMSO) in the absence or presence of p27 siRNA. (A) Increased p27 levels in response to GGTI-2417 and loss of p27 expression and induction in the presence of p27 siRNA. Treated cells were counted via trypan blue exclusion assay to determine inhibition of proliferation (B) and induction of cell death (C). (D to F) MEFs lacking p27 expression are unable to die in response to GGTI-2417. p27 wt and p27 null MEFs were treated with 50 μM GGTI-2417 for 72 h and processed by further assays. (D) GGTI-2417 increases p27 levels in wt, but not null, MEFs and induces pRb hypophosphorylation in both p27 wt and p27 null MEFs. Cells were counted via trypan blue exclusion assay for inhibition of proliferation (E) and induction of cell death (F). The results represent the averages of two independent experiments, each done in triplicates. +, present; −, absent.

GGTI-2418 significantly inhibits the growth of breast tumor xenografts and induces regression of ErbB2-driven mammary tumors in transgenic mice. (A) Nude mice implanted with MDA-MB-231 breast cancer tumors in the mammary fat pads were injected intraperitoneally with either vehicle daily (⧫), 100 mg/kg GGTI-2418 daily (□), or 200 mg/kg GGTI-2418 every third day (▵). (B to C) Effect of GGTI-2418 treatment on mammary tumor progression in ErbB2 transgenic mice. (D) Effect of GGTI-2418 on p27 in vivo. Tumor biopsies were obtained from ErbB2 transgenic mice before and after vehicle or GGTI-2418 treatment and prepared for Western blot analyses of select proteins. As expected, GGTI-2418 accumulates unprenylated Rap1 and prevents the activation of Akt. GGTI-2418 also upregulates p27 levels in vivo. The numbers above the Western blot indicate the fold change in posttreatment samples, as determined by densitometric analysis. The change was 0.63 ± 0.19-fold in vehicle-treated mice and 2.68 ± 0.63-fold in GGTI-2418-treated mice (P = 0.03).