Abstract 3076: Characterization of an improved derivative of the Rac/PAK inhibitor EHop-016 in breast cancer
Authors: Castillo-Pichardo Linette , Humphries-Bickley Tessa , Forrestier-Roman Ingrid , Borrero-Garcia Luis , Pagan Melendez Fabiola , Hernandez Eliud , Vlaar Cornelis , Cubano Luis A. , Dharmawardhane Surangani 
Published: July 2016

The expression and activities of the Rho GTPases Rac and Cdc42, and their downstream effector P21-activated kinase (PAK), have been correlated with metastatic cancer. Our published studies with the Rac/PAK inhibitor EHop-016 demonstrate the validity of using Rac inhibitors as anti metastatic cancer therapeutics. However, the relatively high effective concentrations (Rac activity IC50 1μM, tumor inhibition at 25 mg/kg body weight (BW)), and the moderate bioavailability (∼30%, t1/2 4.5h) of EHop-016 need improvement. Therefore, we developed EHop-016 derivatives and identified EHop-167 as a Rac inhibitor at nM concentrations. Unlike EHop-016, which does not substantially change breast cancer cell shape, but only reduces cell surface actin based invadopodia, EHop-167 induced a marked decrease in breast cancer cell polarity, cell surface extensions, and cell-extracellular matrix (ECM) attachments (focal adhesions). This phenotype of cell rounding and detachment in response to EHop-167 was demonstrated only by breast cancer cell lines that have undergone epithelial to mesenchymal transition, but not by epithelial breast cancer cells, or MCF-10A mammary epithelial cells. As assessed by pulldown assays and western blotting, Rac and PAK activities were reduced by 80-90% in response to 250 nM EHop-167, in the detached cells. As demonstrated by caspase assays, the cell rounding and detachment from the ECM ultimately resulted in anoikis (cell death due to loss of focal adhesions). Accordingly, Transwell assays of mesenchymal breast cancer cells following 250 nM Ehop-167 showed a ∼90% reduction in cell migration in the detached breast cancer cells, and a ∼60% inhibition in the attached cells. EHop-167 also reduced the mammosphere formation efficiency of metastatic cancer cells by 50%, indicating an inhibitory effect on cancer stem cells. To determine the in vivo efficacy of EHop-167, athymic nude mice, bearing mammary fatpad tumors of MDA-MB-435 metastatic cancer cells, were treated 3X a week with 0, 1, or 10 mg/kg BW EHop-167 for 50 days. Treatment with 1.0 mg/kg BW EHop-167 resulted in a 50% reduction in tumor growth, while 10.0 mg/kg BW EHop-167 induced an ∼95% reduction in tumor growth, compared to controls. Additionally, these mice did not show gross signs of toxicity or significant weight loss. Since the parental compound EHop-016 has no anticancer effects at similar concentrations, we conclude that EHop-167 is an improved Rac/PAK inhibitor that holds promise as an anti metastatic breast cancer therapeutic.

Citation Format: Linette Castillo-Pichardo, Tessa Humphries-Bickley, Ingrid Forrestier-Roman, Luis Borrero-Garcia, Fabiola Pagan Melendez, Eliud Hernandez, Cornelis Vlaar, Luis A. Cubano, Surangani Dharmawardhane. Characterization of an improved derivative of the Rac/PAK inhibitor EHop-016 in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3076.