Characterization of a Dual Rac/Cdc42 Inhibitor MBQ-167 in Metastatic Cancer

Abstract

The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42 homolog) regulate cell functions governing cancer malignancy, including cell polarity, migration, and cell-cycle progression. Accordingly, our recently developed Rac inhibitor EHop-016 (IC50, 1,100 nmol/L) inhibits cancer cell migration and viability and reduces tumor growth, metastasis, and angiogenesis in vivo Herein, we describe MBQ-167, which inhibits Rac and Cdc42 with IC50 values of 103 and 78 nmol/L, respectively, in metastatic breast cancer cells. Consequently, MBQ-167 significantly decreases Rac and Cdc42 downstream effector p21-activated kinase (PAK) signaling and the activity of STAT3, without affecting Rho, MAPK, or Akt activities. MBQ-167 also inhibits breast cancer cell migration, viability, and mammosphere formation. Moreover, MBQ-167 affects cancer cells that have undergone epithelial-to-mesenchymal transition by a loss of cell polarity and inhibition of cell surface actin-based extensions to ultimately result in detachment from the substratum. Prolonged incubation (120 hours) in MBQ-167 decreases metastatic cancer cell viability with a GI50 of approximately 130 nmol/L, without affecting noncancer mammary epithelial cells. The loss in cancer cell viability is due to MBQ-167-mediated G2-M cell-cycle arrest and subsequent apoptosis, especially of the detached cells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis in immunocompromised mice by approximately 90%. In conclusion, MBQ-167 is 10× more potent than other currently available Rac/Cdc42 inhibitors and has the potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42. Mol Cancer Ther; 16(5); 805-18. ©2017 AACR.

Conflict of interest statement

Conflict of interest: Authors: E. Hernandez-O-Farrill, C. Vlaar, and S. Dharmawardhane have a conflict of interest due to Provisional patent application: 1,5-disubstituted 1,2,3-triazoles Compounds and Method of Using the Same, Ser. No.: 62/328,282, Filing Date: April 27, 2016

©2017 American Association for Cancer Research.

Figures

Figure 1

Design and synthesis of MBQ-167. Synthesis of MBQ-167. Reaction conditions: (i) conc. H2SO4, NaNO2, water 0–5 °C, 1 h; (ii) NaN3, 0 °C, 1 h, 76%; (iii) THF, 3, 50 °C, 1 h; (iv) NH4Cl (aq), 86%.

Figure 2

Breast cancer cell phenotype following MBQ-167 treatment. A, Upper panel, bright field images of MDA-MB-231 human metastatic breast cancer cells in response to MBQ-167. Lower panel, MCF-7 cells in response to MBQ-167. B,C, Focal adhesions and actin cytoskeleton following MBQ-167 treatment showing representative fluorescence micrographs of MDA-MB-231 cells. MDA-MB-231 cells were treated with 0, 250, or 500 nM MBQ-167 for 24h and fixed and stained in B, with phospho-tyrosine antibodies for focal adhesions (green) and Rhodamine phalloidin for F-actin (red) and in C, with anti vinculin (red). Arrows, invadopodia; arrowheads, focal adhesions.

Figure 3

Inhibitory effect of MBQ-167 on Rac and Cdc42 activation. MDA-MB-231 human breast cancer cells were treated for 24 h with 250 nM MBQ-167. A–C, The attached (A) and detached (D) cell populations were recovered and equal amounts of proteins subjected to pulldown assays using the p21-binding domain of PAK to isolate the GTP bound Rac and Cdc42. Cell lysates were western blotted with antibodies to Rac or Cdc42. Results from positive bands in western blots were quantified using image J. A, Left, Representative western blot for Rac1/2/; right, quantification of Rac activation at 24 h following 0 or 250nM MBQ-167. B, Left, Representative western blot for Cdc42; right, quantification of Cdc42 activation following 24 h treatment with 0 or 250 nM MBQ-167. The integrated density for active Rac or Cdc42 (GTP) was divided by the total Rac or Cdc42 from the same cell lysates. Rac or Cdc42 activity for each MBQ-167 treatment was divided by the vehicle controls for each experiment to obtain Relative Rac or Cdc42 activity. N=3, * = P < 0.05, *** = P < 0.001. Error bars represent ± S.E.M. C,D, MDA-MB-231 cells with vehicle control (0.1% DMSO) or varying concentrations of MBQ-167 (0–1000 nM) were treated for 24 hrs. Total cell lysates using combined attached and detached treated populations were subjected to the G-LISA Rac1/2/3 or Cdc42 activation assay. IC50 curves for percentage Rac (C) or Cdc42 (D) activation are relative to vehicle from three biological replicates each with two technical replicates. Error bars represent ± S.D. Four-parameter dose-response curves generated using GraphPad Prism® are shown.

Figure 4

The effect of MBQ-167 on signaling downstream of Rac and Cdc42. A, The effect of MBQ-167 on PAK1 and PAK2 phosphorylation as measured by western blotting for pPAK1 (T423)/pPAK2 (T402), pPAK1 (S199)/pPAK2 (S192), and pPAK1 (S144) levels in MDA-MB-231 cells after 24 hours of treatment in 0 or 250 nM MBQ-167. Data for separate attached (Att) and detached (Det) populations are shown. Left, representative western blots (N=3). Right, relative PAK activity following MBQ-167 treatment. Positive bands from all western blots were quantified using image J. The integrated density of p-PAK was divided by that of total PAK for the same cell lysate and used as a measure of PAK activity for each phospho PAK residue. Relative PAK activity was determined relative to vehicle controls for each experiment. N=3, * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Error bars represent ± S.E.M. B, Effect of MBQ-167 on PAK downstream effectors LIMK and cofilin phosphorylation. MDA-MB-231 cells were incubated for 4, 12, or 24 h in vehicle or 250 nM MBQ-167, the attached (A) and detached (D) populations were separated, lysed, and equal protein used for western blotting. Representative western blot of total LIMK1 or p-LIMK1/2 (Y507/T508) following 24 h in 0 or 250 nM MBQ-167 (N=2) is shown. C, Representative western blot of total or p-cofilin (S3) of equal amounts of total protein lysates following 4, 12, or 24 h in 250nM MBQ-167 (N=3). Separated attached (A) and detached (D) populations are shown for 12 and 24h of MBQ-167 treatment. D, Effect of MBQ-167 on STAT3 phosphorylation and expression. Representative western blot is shown for pSTAT3 (Y705) and total STAT3 expression in GFP-HER2-BM cells after 24h treatment with vehicle or 100, 200, or 500 nM MBQ-167. Representative western blot (left) and quantification (right). N=3, * = P< 0.05, Error bars represent ± S.E.M. E, F, Effect of MBQ-167 on cell migration. E, The effect of MBQ-167 on MDA-MB-231 cellular migration as measured by a transwell assay. Images are representative of three independent experiments. The Graph below shows quantification of 20 microscopic fields per treatment per experiment of PI stained cells that migrated to the underside of the membrane through 8 micron diameter pores N=3,* = P < 0.05, Error bars represent ± S.E.M. F, The effect of MBQ-167 on cell migration in a scratch assay. MDA-MB-231 cells plated at equal density were subjected to a scratch in the center and treated with MBQ-167 at 0, 250, or 500 nM. Micrographs were digitally acquired at 0 and 24 h and the distance of the scratch quantified for each treatment and presented relative to the distance at time 0. Results are an average of two technical replicates and two biological replicates for each treatment ± S.D,* = P < 0.05. G, The effect of MBQ-167 on mammosphere forming efficiency in MDA-MB-231 cells. MDA-MB-231 cells treated with 0 or 250 nM MBQ-167 were subjected to mammosphere assays for 4 days. Cells were treated with MBQ-167 only once prior to placing on the mammosphere medium. Mammosphere forming efficiency was calculated as the percentage of the number of mammospheres divided by the number of cells seeded per well. N=3, *** = P < 0.001, Error bars represent ± S.D.

Figure 5. Effect of MBQ-167 on cell survival

A, The effect of MBQ-167 on MDA-MB-231, GFP-HER2-BM, and MCF10A cell viability. Equal numbers of each respective cell line were treated with vehicle control (0.1% DMSO) or varying concentrations of MBQ-167 (0–1000 nM) for 120 h. GI50 curves for percentage cell viability are relative to vehicle from three biological replicates each with two technical replicates. Four-parameter dose-response curves generated using GraphPad Prism® are shown. N=3, Error bars represent ± S.E.M. B, The effect of MBQ-167 on cell cycle progression. Equal numbers of MDA-MB-231 cells in either vehicle control or treatment groups were treated for 24 h with 0 or 250 nM MBQ-167. Graphs represent the percentage of control versus 250 nM MBQ-167 treated cells stained with PI in G0/G1, S, or G2/M phases of the cell cycle. Left graph, representative flow cytometry analysis; right graph, quantification of cell cycle stage. N=3, Error bars ± S.E.M. C, The effect of MBQ-167 on caspase3,-7 activity. Left graph, caspase3/7 activity of MDA-MB-231 and GFP-HER2-BM cells (including attached and detached populations) following vehicle (0.1% DMSO) or varying concentrations of MBQ-167 (0–1000 nM) for 24 h. N=3, Error bars represent ± S.E.M. Right graph, the effect of MBQ-167 on caspase-3,-7 activity of MDA-MB-231 cells. Cells were treated with 250 nM MBQ-167 for 24 h and equal numbers of separated attached and detached cells were lysed and used for caspase 3/7 assays. Caspase-3,-7 activity relative to equal number of attached cells from control cells is shown. N=3, Error bars represent ± S.E.M. D, The effect of MBQ-167 on mitochondrial regulation of apoptosis. The effect of MBQ-167 on the expression of the pro-survival proteins Bcl-2, Bcl-xL, and Mcl-1 in MDA-MB-231 cells after 24h of treatment. Left panel, representative western blot; Right panel, quantification of the integrated density of positive bands using image J. N=3, * = P < 0.05, *** = P < 0.001, Error bars represent ± S.E.M.

Figure 6

In-vivo efficacy of MBQ-167 compared with EHop-016. Mammary fatpad tumors were established in nude mice by inoculating 5 × 105 GFP-HER2-BM cells. Following one week, mice were treated with vehicle control or 1.0, or 10.0 mg/kg body weight (BW) MBQ-167 3X a week by i.p. injection. A, Left panel, representative excised tumors following 0, 1, 10 mg/kg BW MBQ-167. Right panel, average relative tumor growth from fluorescence in situ images up to 65 days following 0, 1.0, or 10 mg/kg BW MBQ-167 (3X a wk) (N=6). B, Representative fluorescence micrographs of lungs, spleens, and kidneys from vehicle or MBQ-167 treated mice following necropsy. C, Mouse weights from 1–65 days. D, E, Liver enzyme activities following MBQ-167 treatment. Following necropsy, livers were harvested, lysed and subjected to D, ALP activity or E, ALT activity assays. N=4, error bars represent ± S.E.M.