Inhibition of Cdc42-intersectin interaction by small molecule ZCL367 impedes cancer cell cycle progression, proliferation, migration, and tumor growth

Abstract

Cdc42 is a member of the Rho family of small GTPases that are at the crossroads of major oncogenic signaling pathways involved in both lung and prostate cancers. However, the therapeutic potential of Cdc42 regulation is still unclear due to the lack of pharmacological tools. Herein, we report that ZCL367 is a bona fide Cdc42 inhibitor that suppressed cancer development and ZCL278 can act as a partial Cdc42 agonist. In lung cancer cell lines with varying EGFR and Ras mutations as well as both androgen-independent and androgen-dependent prostate cancer cell lines, ZCL367 impeded cell cycle progression, reduced proliferation, and suppressed migration. ZCL367 decreased Cdc42-intersectin interactions and reduced Cdc42-mediated filopodia formation. ZCL367 showed increased potency and selectivity for Cdc42 when compared to Rac1 and RhoA. ZCL367 reduced A549 tumorigenesis in a xenograft mouse model. Altogether, ZCL367 is a selective Cdc42 inhibitor and an excellent candidate for lead compound optimization for further anticancer studies.

Keywords: Cdc42; Rho GTPase; drug development; intersectin; lung cancer; prostate cancer.

Figure 1.

ZCL compound screening and validation as Cdc42-ITSN regulators. (a) ZCL367 inhibits migration/wound healing of A549 and PC3 cells. Confluent cells were scratched and treated with mitomycin C before treating with ZCL compound. The percentage of the wound closure was quantified from three scratches from two independent experiments and is expressed as mean±SEM. (b) Molecular docking of ZCL278 (green) and ZCL367 (pink) into the Cdc42-ITSN binding site. ZCL278 and ZCL367 show different poses with a certain extent of overlap with each other. ZCL278 formed two hydrogen bonds with Thr35 and Asp57, and hydrophobic interactions with Val36 and Thr35 of Cdc42. ZCL367 formed three hydrogen bonds with Asp38, Asn39, and Asp57, and hydrophobic interactions with Phe56 and Val36 of Cdc42. Gray: Cdc42, light purple: ITSN, orange dots: hydrogen bond. (c) ZCL compounds inhibit GEF-mediated Rho GTPase activation. Both ZCL278 (IC₅₀ = 7.5 μM) and ZCL367 (IC₅₀ = 0.098 μM) inhibit DH domain-mediated mant-GTP binding/Cdc42 activation. ZCL367 is more potent against Cdc42 than RhoA (IC₅₀ = 29.7 μM) and Rac1 (IC₅₀ = 0.19 μM). (d) ZCL278 activates Cdc42. Serum-starved Swiss 3T3 cells were treated with ZCL278 and analyzed for Cdc42 and Rac1 activation via GLISA. (e) ZCL278 increases intrinsic GTP binding of Cdc42. In the absence of GEF/DH domain, treatment with ZCL278 increased binding of GTP to Cdc42. (f) Cdc42 regulators inhibit Cdc42-ITSN. A co-immunoprecipitation of ITSN with active-Cdc42 revealed that ZCL367 (50 μM) and AZA1 (10 μM) was able to dislodge ITSN from active-Cdc42. At the same concentration, ZCL278 was not effective. All data are presented as mean±SEM from duplicates or triplicates from two independent experiments. ANOVA compared treatments to their respective control (* p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001).

Figure 2.

ZCL compounds regulate microspike formation. Swiss 3T3 cells were starved ON then preincubated with ZCL367 (50 μM) for 2 h. Bradykinin (100 ng/mL) was used to activate Cdc42/Rac1 for 20 m. Cells were also treated with ZCL278 (50 μM) for comparison. Cells were fixed and stained with phalloidin. Boxes are magnifications of membrane ruffling and highlighted by →. Circles are magnifications of microspikes and highlighted by ▲. Asterisks (*) highlight diminished stress fibers.

Figure 3.

ZCL367 inhibits Cdc42 activation downstream of GM130. Swiss 3T3 cells were starved ON then preincubated with ZCL367 (50 μM) for 2 h. Bradykinin (100 ng/mL) was used to activate Cdc42/Rac1 for 20 m. Cells were fixed and stained for GM130 (red), nucleus (blue), and phalloidin (green). GM130 organization was conserved in ZCL367 treated cells. Activation with bradykinin resulted in altered distribution of GM130. Treatment with ZCL367 inhibited filopodia formation but did not affect GM130 distribution. Cells that showed strong Rac1 activation in the presence of ZCL367 showed a GM130 staining similar to control.

Figure 4.

Cdc42 inhibition alters critical cellular processes. (a) Cdc42 regulators decrease cell proliferation. Relative cellular density of lung and prostate cancer cells up to 144 h after treatment with ZCL278, ZCL367, or AZA1. (b) Cdc42 inhibitors alter FBS-stimulated cell cycle progression. Relative cell cycle distribution of lung and prostate cancer cells after 24 h of starvation followed by 24 h FBS-stimulation with or without treatment (A549, HCC827, H522 = 50 μM ZCL367 or 10 μM AZA1; PC3, DU145, 22Rv1, LNCaP = 20 μM ZCL367 or 5 μM AZA1). (c) Effect of Cdc42 inhibitors on cell viability. Cells (5 × 10⁴)  were seeded and, after 24 h, treated with compound for 24 h. A solution of MTT was added and the reading was taken after 3 h. 50 μM NSC23766 (NSC), a Rac1 inhibitor, and 50 μM NSC + 50 μM ZCL367 is not as toxic as AZA1 alone. The absorbance was measured at 562 nm. All data are presented as mean±SEM from duplicates or triplicates from two independent experiments. ANOVA compared treatments to their respective control (* p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001).

Figure 5.

ZCL367 inhibits A549 tumor growth in mice. Mice with palpable A549 tumors were treated with ZCL367 (20 μg/g) or vehicle control (10% DMSO in sesame oil) every other day for 28 days. Tumor volume was calculated using the formula (length x width²)/2. After 28 days, the tumor was weighed. Data are presented as mean±SEM from groups of n = 6. ANOVA followed by Matt–Whitney test compared ZCL367 treatment to control (* p < 0.05).