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. 2014;14(6):840-51.
doi: 10.2174/18715206113136660334.

Preclinical development of novel Rac1-GEF signaling inhibitors using a rational design approach in highly aggressive breast cancer cell lines

Georgina A Cardama, Maria J Comin, Leandro Hornos, Nazareno Gonzalez, Lucas Defelipe, Adrian G Turjanski, Daniel F Alonso, Daniel E Gomez, Pablo Lorenzano Menna  1
Affiliations
Free PMC article

Preclinical development of novel Rac1-GEF signaling inhibitors using a rational design approach in highly aggressive breast cancer cell lines

Georgina A Cardama et al. Anticancer Agents Med Chem. 2014.
Free PMC article

Abstract

Rho GTPases play a key role in the regulation of multiple essential cellular processes, including actin dynamics, gene transcription and cell cycle progression. Aberrant activation of Rac1, a member of Rho family of small GTPases, is associated with tumorigenesis, cancer progression, invasion and metastasis. Particularly, Rac1 is overexpressed and hyperactivated in highly aggressive breast cancer. Thus, Rac1 appears to be a promising and relevant target for the development of novel anticancer drugs. We identified the novel Rac1 inhibitor ZINC69391 through a docking-based virtual library screening targeting Rac1 activation by GEFs. This compound was able to block Rac1 interaction with its GEF Tiam1, prevented EGF-induced Rac1 activation and inhibited cell proliferation, cell migration and cell cycle progression in highly aggressive breast cancer cell lines. Moreover, ZINC69391 showed an in vivo antimetastatic effect in a syngeneic animal model. We further developed the novel analog 1A-116 by rational design and showed to be specific and more potent than the parental compound in vitro and interfered Rac1-P-Rex1 interaction. We also showed an enhanced in vivo potency of 1A-116 analog. These results show that we have developed novel Rac1 inhibitors that may be used as a novel anticancer therapy.

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Figures

Fig. (1)
Fig. (1)
ZINC69391 is able to block Rac1 activation in breast cancer cells by interference of Rac1-Tiam1 interaction. A, Chemical structure of ZINC69391 (C14H15F3N5; molecular weight, 310.303) B, Concentration-dependent blockade of Rac1-Tiam1 interaction. Constitutive active Tiam1-HA tagged expressed in HEK-293T cells was affinity-precipitated with bacterially expressed Rac1 immobilized in Glutathione Agarose Beads in the presence of varying concentrations (100 and 200μM) of ZINC69391. Western blot analysis was carried out with antibodies for HA tag. The experiment was repeated 3 times. C, Densitometric analysis on the Western blot showed in B ***, p<0.001 determined by ANOVA cont. Dunnett´s Multiple Comparison Test. Each bar represents the media ± SD considering the results of three independent experiments D, Concentration-dependent Rac1 inhibition by ZINC69391 in F3II cells. Serum-starved F3II cells were treated for 1 hour at different ZINC69391 concentrations and stimulated with EGF (100ng/ml) for 15 minutes. E, Cdc42 was not affected by ZINC69391 treatment. F3II cells were treated in full growth media conditions.
Fig. (2)
Fig. (2)
ZINC69391 affects cell viability and cell cycle progression of aggressive breast cancer cells. A, ZINC69391 inhibits cell proliferation. Breast cancer cell lines F3II (●), MDA-MB-231 (■) and MCF7 (▲) were treated for 72 hours with different concentrations of ZINC69391. Cell viability was measured using MTT assay. B and C, ZINC69391 arrest cell cycle progression in G1 phase. MDA-MB-231 cells were synchronized and treated for 48 hours with ZINC69391 10μM. Cells were fixed, stained with propidium iodide and analyzed by FACS to estimate the percentage of cells in G1 phase, S phase and G2/M phase. Columns, mean of a representative experiment (n=3) of three independent experiments; bars, SD. *p<0.05. ***,p<0.001 determined by Student's t test versus control in each phase.
Fig. (3)
Fig. (3)
ZINC69391 blocks actin cytoskeleton reorganization and inhibits cell migration. A, Representative micrographs taken at 1000X showing inhibition of EGF-induced actin reorganization by ZINC69391 in F3II and MDA-MB-231 cells. Cells were grown on coverslips, serum-starved for 16 hs (untreated panel) and treated for 1 hour with ZINC69391 10 μM and 50 μM. After 15 minutes stimulation with EGF (100 ng/ml) (EGF control), cells were fixed and actin filaments were visualized with AlexaFluor555-phalloidin. B, Confluent monolayers of F3II cells were scratched and treated with ZINC69391 10 μM and 50 μM in presence of FBS. After 16 hours incubation, cells were fixed and stained. Wound closure was analyzed using NISElement 3.0. Experiments were carried out in triplicate plates. Results are expressed as percentage of wound closure, expressed as mean (n = 10). Bars, S.E.M. ***, p<0.001. ANOVA cont. Dunnett´s Multiple comparison test C, The same experimental design was carried out with MDA-MB-231 cells. Results are expressed as percentage of wound closure, and expressed as mean (n = 10), bars S.E.M. **, p<0.01; ***, p<0.001. ANOVA cont. Dunnett´s Multiple Comparison Test.
Fig. (4)
Fig. (4)
Anti-metastatic effect of ZINC69391 on F3II cells. A, 2x105 viable F3II cells were injected into the lateral tail vein and the mice were treated i.p with a daily dose of 25 mg/kg. On day 21 mice were sacrificed and superficial lung nodules were counted. Each data point represents the number of total lung nodules per mouse in each treatment group (n=10 control group, n=8 treated group) **, p < 0.01. Mann-Whitney test. B, Experimental formation of lung macronodules (>1 mm in diameter). **p < 0.01 Mann-Whitney Test C, Representative left lung lobes were photographed.
Fig. (5)
Fig. (5)
Best docking poses of ZINC69391 and its derived novel analog 1A-116 over Rac1. A Chemical structure of 1A-116 (C16H16F3N3; molecular weight, 307.31) B, Cartoon representation of the structure of Rac1 showing the binding site defined by Trp56. C, The binding pocket of ZINC69391 (Drawn with ball and sticks representation) depicting key interactions with Rac1 (Structure drawn in cartoon and residues drawn in Licorice representation). D, Rac1 surface with 1A-116 docking pose (Drawn in ball and sticks).
Fig. (6)
Fig. (6)
Expression of Rac1-G12V constitutively active variant attenuates the effect of 1A-116. MDA-MB-231 were transfected with Rac1-G12V variant or empty vector (EV). These cells were treated for 48 hs with different concentrations of 1A-116 analog. The constitutively active Rac1 variant confers GEF independence, showing an attenuated effect of 1A116 analog compared to the control cells. Bars SD, ***p<0.001. EV vs. Rac1 G12V at each concentration. Two way ANOVA cont. Bonferroni Test.
Fig. (7)
Fig. (7)
1A-116 interferes P-Rex1-Rac1 interaction, being a more potent Rac1 inhibitor than ZINC69391. A, Affinity precipitation showing that 1A-116 is capable of blocking P-Rex1-Rac1 interaction. B, F3II cells were treated for 12 hour with ZINC69391 and 1A-116 in full growth conditions. Pull down assays were carried out and then were analyzed by Western blot. 1A-116 markedly reduced Rac1-GTP levels compared to ZINC69391 C, 1A-116 had no effect on Cdc42 activation.
Fig. (8)
Fig. (8)
Effect of 1A-116 treatment on experimental lung tumor formation. F3II cells were injected intravenously in mice (n=10), as described in “Materials and Methods.” A, Total lung tumor nodules experimentally formed. The control group was treated with vehicle and the second group of animals was treated with 1A-116 analog with a 3 mg/kg body weight/day dose. *p < 0.05 Mann-Whitney Test. B, Experimental formation of lung macronodules (>1 mm in diameter). *p < 0.05 Mann-Whitney Test. C, Total weight of lungs corresponding to both groups. The continuous line represents the average pulmonary weight of Balb/c mice described by Han et al., [46]. **p<0.01 Mann-Whitney Test.
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