The objective of this project is to design novel therapeutics targeting cancer metastasis, the most deadly aspect of epithelial cancers. Our previous studies have shown that Rac, a key signaling intermediate that regulates actin cytoskeletal changes during cell migration/invasion and cancer metastasis, is associated with high cancer metastatic efficiency. Since Rac proteins are activated by GDP/GTP exchange, inhibition of the interaction of Rac with its guanine nucleotide exchange factors (GEF) is expected to inhibit Rac activity. Since the known Rac inhibitor NSC-23766 is not effective at therapeutically useful concentrations, we synthesized novel NSC-23766 derivatives. Results show that the new inhibitor EHop-016 is 100-fold more efficient than the parent compound at inhibiting Rac activity of metastatic cancer cells without affecting the activity of the related GTPase, Rho. EHop-016 inhibited Rac activity of metastatic cancer cells with an IC50 of 0.78 microM. Cell viability assays, performed to test for the toxicity of this compound, demonstrated that at concentrations below 5 microM, EHop-016 did not affect mammary epithelial cell (MCF-10A) viability and decreased proliferation of metastatic cancer cells (MDA-MB-435) by only 20%. We conclude that EHop-16 is a potent Rac inhibitor with promise of further development as a small molecule inhibitor of cancer cell invasion, and thus, metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3251. doi:10.1158/1538-7445.AM2011-3251

Background The significant challenge in treating triple-negative breast cancer (TNBC) lies in its high rate of distant metastasis. To address this, inhibiting metastasis formation in TNBC is vital. Rac is a key player in cancer metastasis. Previously, we developed Ehop-016, a Rac inhibitor that successfully reduced tumor growth and metastasis in mice. In this study, we assessed the effectiveness of HV-107, a derivative of Ehop-016, in inhibiting TNBC metastasis at lower doses. Methods Rho GTPases activity assays were performed with the use of GST-PAK beads and Rac, Rho, and Cdc42 GLISA. Cell viability was assessed through trypan blue exclusion and MTT assays. Cell cycle analysis was conducted using flow cytometry. To evaluate invading capabilities, transwell assays and invadopodia formation assays were performed. Metastasis formation studies were conducted using a breast cancer xenograft mouse model. Results HV-107 inhibited Rac activity by 50% in MDA-MB-231 and MDA-MB-468 cells at concentrations of 250–2000 nM, leading to a 90% decrease in invasion and invadopodia activity. Concentrations of 500 nM and above caused dose-dependent reductions in cell viability, resulting in up to 20% cell death after 72 h. Concentrations exceeding 1000 nM upregulated PAK1, PAK2, FAK, Pyk2, Cdc42, and Rho signallings, while Pyk2 was downregulated at 100–500 nM. Through in vitro experiments, optimal concentrations of HV-107 ranging from 250 to 500 nM were identified, effectively inhibiting Rac activity and invasion while minimizing off-target effects. In a breast cancer xenograft model, administration of 5 mg/kg HV-107 (administered intraperitoneally, 5 days a week) reduced Rac activity by 20% in tumors and decreased metastasis by 50% in the lungs and liver. No observed toxicity was noted at the tested doses. Conclusion The findings indicate that HV-107 exhibits promising potential as a therapeutic medication utilizing Rac inhibition mechanisms to address metastasis formation in TNBC.