A novel geranylgeranyl transferase inhibitor in combination with lovastatin inhibits proliferation and induces autophagy in STS-26T MPNST cells

Abstract

Prenylation inhibitors have gained increasing attention as potential therapeutics for cancer. Initial work focused on inhibitors of farnesylation, but more recently geranylgeranyl transferase inhibitors (GGTIs) have begun to be evaluated for their potential antitumor activity in vitro and in vivo. In this study, we have developed a nonpeptidomimetic GGTI, termed GGTI-2Z [(5-nitrofuran-2-yl)methyl-(2Z,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenyl 4-chlorobutyl(methyl)phosphoramidate], which in combination with lovastatin inhibits geranylgeranyl transferase I (GGTase I) and GGTase II/RabGGTase, without affecting farnesylation. The combination treatment results in a G(0)/G(1) arrest and synergistic inhibition of proliferation of cultured STS-26T malignant peripheral nerve sheath tumor cells. We also show that the antiproliferative activity of drugs in combination occurs in the context of autophagy. The combination treatment also induces autophagy in the MCF10.DCIS model of human breast ductal carcinoma in situ and in 1c1c7 murine hepatoma cells, where it also reduces proliferation. At the same time, there is no detectable toxicity in normal immortalized Schwann cells. These studies establish GGTI-2Z as a novel geranylgeranyl pyrophosphate derivative that may work through a new mechanism involving the induction of autophagy and, in combination with lovastatin, may serve as a valuable paradigm for developing more effective strategies in this class of antitumor therapeutics.

Fig. 1.

Synthesis of prodrug GGTI-2Z (compound 7) and structures of 2Z-GGMP (compound 8) and 2Z-GGPP (compound 9). TEA, triethylamine; DCM, dichloromethane.

Fig. 2.

Inhibition of prenylation in STS-26T cells by GGTI-2Z/lovastatin combination treatment. STS-26T cultures were treated as indicated for 24 or 48 h. Whole-cell lysates were probed for prenylation status of Ras superfamily GTPases via Western analysis. A, detection of Rap1A via an antibody directed toward the unprenylated form of Rap1A and detection of total Rap1. B, detection of RhoA, which has been reported to be overexpressed after block of GGTase. C, detection of Rab5. Unprenylated GTPases migrate more slowly on SDS-polyacrylamide gel electrophoresis gels. β-Tubulin was used as a loading control in all Western blots. Results shown are representative of at least three independent experiments.

Fig. 3.

Lack of effect of GGTI-2Z/lovastatin treatment on membrane localization of a farnesylated GFP construct. HEK293 cells were transiently transfected with pRK7.GFP.H-Ras.CaaX plasmid, followed by treatment with prenylation inhibitors as shown for 16 h. FTI-1/lovastatin treatment inhibited H-Ras.CaaX localization at the plasma membrane, whereas GGTI-2Z/lovastatin treatment did not affect the localization even at 6 μM GGTI-2Z concentration. Results are representative of three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 4.

A and B, inhibition of proliferation of STS-26T cells after GGTI-2Z/lovastatin treatment. Samples were collected every 24 h after treatment for analysis of cell number. Data represent means ± S.D. of three independent experiments. C, number of nonviable cells was analyzed with respect to total number of cells to calculate percentage of viability at the given time points. D, cells were treated with the compounds at several different concentrations as shown, alone, or in combination, and the data were tested for synergy using isobologram analysis. Data represent means ± S.D. of two independent experiments.

Fig. 5.

GGTI-2Z/lovastatin treatment arrests STS-26T cells in G₀/G₁. STS-26T cells were treated as shown. Cultures were harvested 48 h after treatment for DNA content by staining with propidium iodide. Histograms represent 10⁴ events, and the cell-cycle profile was determined by using Modfit. Results are representative of two independent experiments.

Fig. 6.

Lack of apoptosis in STS-26T cells after GGTI-2Z/lovastatin treatment. HA14-1-treated cells were used as a positive control. A, STS-26T cells were treated as indicated. Data represent means of triplicate samples and are representative of two independent experiments. B, STS-26T cells were treated as indicated. Attached and detached cells were pooled, and whole-cell lysates were separated and probed for cleaved caspase-3. C, STS-26T cells were treated for 48 h with the indicated concentrations of lovastatin and/or GGTI-2Z or for 30 min with HA14-1. Nuclei were stained with Hoechst 33342, and live-cell imaging was performed on a LSM-510 microscope at 40× magnification.

Fig. 7.

Induction of autophagy in STS-26T cells by GGTI-2Z/lovastatin treatment. STS-26T cells were treated with the indicated concentrations of GGTI-2Z and lovastatin alone or in combination. Results are representative of three independent experiments. A, treatments were for 24 or 48 h as shown. Whole-cell lysates were then probed for LC3 and β-tubulin. B, cells were subject to 2-h pretreatment with protease inhibitors, 10 μM pepstatin A, and 10 μM E64D as indicated. Whole-cell lysates were then probed for LC3 and β-tubulin. C, cells were subject to 48 h of drug treatment, with addition of 50 nM bafilomycin A1 for the last 2 h of the incubation as indicated. Whole-cell lysates were then probed for LC3 and β-tubulin. D, STS-26T cells were treated as indicated for 48 h followed by methanol fixation. Cells were then stained for LC3 and LAMP-2. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and cells were visualized under a LSM-510 microscope at 40× magnification. E, quantitative analysis of LC3-positive puncta treated with either DMSO or the drug combination for 48 h.