Characterization of Novel Derivatives of MBQ-167, an inhibitor of the GTP-binding proteins Rac/Cdc42.

Rac and Cdc42, are homologous GTPases that regulate cell migration, invasion, and cell cycle progression; thus, representing key targets for metastasis therapy. We previously reported on the efficacy of MBQ-167, which blocks both Rac1 and Cdc42 in breast cancer cells and mouse models of metastasis. To identify compounds with increased activity, a panel of MBQ-167 derivatives was synthesized, maintaining its 9-ethyl-3-(1H-1,2,3-triazol-1-yl)-9H-carbazole core. Similar to MBQ-167, MBQ-168 and EHop-097, inhibit activation of Rac and Rac1B splice variant and breast cancer cell viability, and induce apoptosis. MBQ-167 and MBQ-168 inhibit Rac and Cdc42 by interfering with guanine nucleotide binding, and MBQ-168 is a more effective inhibitor of PAK (1,2,3) activation. EHop-097 acts via a different mechanism by inhibiting the interaction of the guanine nucleotide exchange factor (GEF) Vav with Rac. MBQ-168 and EHop-097 inhibit metastatic breast cancer cell migration, and MBQ-168 promotes loss of cancer cell polarity to result in disorganization of the actin cytoskeleton and detachment from the substratum. In lung cancer cells, MBQ-168 is more effective than MBQ-167 or EHop-097 at reducing ruffle formation in response to EGF. Comparable to MBQ-167, MBQ-168 significantly inhibits HER2+ tumor growth and metastasis to lung, liver, and spleen. Both MBQ-167 and MBQ-168 inhibit the cytochrome P450 (CYP) enzymes 3A4, 2C9, and 2C19. However, MBQ-168 is ~10X less potent than MBQ-167 at inhibiting CYP3A4, thus demonstrating its utility in relevant combination therapies. In conclusion, the MBQ-167 derivatives MBQ-168 and EHop-097 are additional promising anti metastatic cancer compounds with similar and distinct mechanisms.

Efficacy of Rac and Cdc42 Inhibitor MBQ-167 in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer, with a high predisposition for locally invasive and metastatic cancer. With the objective to reduce cancer metastasis, we developed small molecule inhibitors to target the drivers of metastasis, the Rho GTPases Rac and Cdc42. Of these, MBQ-167 inhibits both Rac and Cdc42 with IC50s of 103 and 78 nmol/L, respectively; and consequently, inhibits p21-activated kinase (PAK) signaling, metastatic cancer cell proliferation, migration, and mammosphere growth; induces cell-cycle arrest and apoptosis; and decreases HER2-type mammary fatpad tumor growth and metastasis (Humphries-Bickley and colleagues, 2017). Herein, we used nuclear magnetic resonance to show that MBQ-167 directly interacts with Rac1 to displace specific amino acids, and consequently inhibits Rac.GTP loading and viability in TNBC cell lines. Phosphokinome arrays in the MDA-MB-231 human TNBC cells show that phosphorylation status of kinases independent of the Rac/Cdc42/PAK pathway are not significantly changed following 200 nmol/L MBQ-167 treatment. Western blotting shows that initial increases in phospho-c-Jun and phospho-CREB in response to MBQ-167 are not sustained with prolonged exposure, as also confirmed by a decrease in their transcriptional targets. MBQ-167 inhibits tumor growth, and spontaneous and experimental metastasis in immunocompromised (human TNBC) and immunocompetent (mouse TNBC) models. Moreover, per oral administration of MBQ-167 at 100 mg/kg body weight is not toxic to immunocompetent BALB/c mice and has a half-life of 4.6 hours in plasma. These results highlight the specificity, potency, and bioavailability of MBQ-167, and support its clinical potential as a TNBC therapeutic.

Rac inhibition as a novel therapeutic strategy for EGFR/HER2 targeted therapy resistant breast cancer.

BACKGROUND: Even though targeted therapies are available for cancers expressing oncogenic epidermal growth receptor (EGFR) and (or) human EGFR2 (HER2), acquired or intrinsic resistance often confounds therapy success. Common mechanisms of therapy resistance involve activating receptor point mutations and (or) upregulation of signaling downstream of EGFR/HER2 to Akt and (or) mitogen activated protein kinase (MAPK) pathways. However, additional pathways of resistance may exist thus, confounding successful therapy. METHODS: To determine novel mechanisms of EGFR/HER2 therapy resistance in breast cancer, gefitinib or lapatinib resistant variants were created from SKBR3 breast cancer cells. Syngenic therapy sensitive and resistant SKBR3 variants were characterized for mechanisms of resistance by mammosphere assays, viability assays, and western blotting for total and phospho proteins. RESULTS: Gefitinib and lapatinib treatments reduced mammosphere formation in the sensitive cells, but not in the therapy resistant variants, indicating enhanced mesenchymal and cancer stem cell-like characteristics in therapy resistant cells. The therapy resistant variants did not show significant changes in known therapy resistant pathways of AKT and MAPK activities downstream of EGFR/HER2. However, these cells exhibited elevated expression and activation of the small GTPase Rac, which is a pivotal intermediate of GFR signaling in EMT and metastasis. Therefore, the potential of the Rac inhibitors EHop-016 and MBQ-167 to overcome therapy resistance was tested, and found to inhibit viability and induce apoptosis of therapy resistant cells. CONCLUSIONS: Rac inhibition may represent a viable strategy for treatment of EGFR/HER2 targeted therapy resistant breast cancer.

Positron Emission Tomography Imaging of Functional Transforming Growth Factor ß (TGFß) Activity and Benefit of TGFß Inhibition in Irradiated Intracranial Tumors.

PURPOSE: Transforming growth factor ß (TGFß) promotes cell survival by endorsing DNA damage repair and mediates an immunosuppressive tumor microenvironment. Thus, TGFß activation in response to radiation therapy is potentially targetable because it opposes therapeutic control. Strategies to assess this potential in the clinic are needed. METHODS AND MATERIALS: We evaluated positron emission tomography (PET) to image 89Zr -fresolimumab, a humanized TGFß neutralizing monoclonal antibody, as a means to detect TGFß activation in intracranial tumor models. Pathway activity of TGFß was validated by immunodetection of phosphorylated SMAD2 and the TGFß target, tenascin. The contribution of TGFß to radiation response was assessed by Kaplan-Meier survival analysis of mice bearing intracranial murine tumor models GL261 and SB28 glioblastoma and brain-adapted 4T1 breast cancer (4T1-BrA) treated with TGFß neutralizing monoclonal antibody, 1D11, and/or focal radiation (10 Gy). RESULTS: 89Zr-fresolimumab PET imaging detected engineered, physiological, and radiation-induced TGFß activation, which was confirmed by immunostaining of biological markers. GL261 glioblastoma tumors had a greater PET signal compared with similar-sized SB28 glioblastoma tumors, whereas the widespread PET signal of 4T1-BrA intracranial tumors was consistent with their highly dispersed histologic distribution. Survival of mice bearing intracranial tumors treated with 1D11 neutralizing antibody alone was similar to that of mice treated with control antibody, whereas 1D11 improved survival when given in combination with focal radiation. The extent of survival benefit of a combination of radiation and 1D11 was associated with the degree of TGFß activity detected by PET. CONCLUSIONS: This study demonstrates that 89Zr-fresolimumab PET imaging detects radiation-induced TGFß activation in tumors. Functional imaging indicated a range of TGFß activity in intracranial tumors, but TGFß blockade provided survival benefit only in the context of radiation treatment. This study provides further evidence that radiation-induced TGFß activity opposes therapeutic response to radiation.

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

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.

Equol, an isoflavone metabolite, regulates cancer cell viability and protein synthesis initiation via c-Myc and eIF4G.

Epidemiological studies implicate dietary soy isoflavones as breast cancer preventives, especially due to their anti-estrogenic properties. However, soy isoflavones may also have a role in promoting breast cancer, which has yet to be clarified. We previously reported that equol, a metabolite of the soy isoflavone daidzein, may advance breast cancer potential via up-regulation of the eukaryotic initiation factor 4GI (eIF4GI). In estrogen receptor negative (ER-) metastatic breast cancer cells, equol induced elevated levels of eIF4G, which were associated with increased cell viability and the selective translation of mRNAs that use non-canonical means of initiation, including internal ribosome entry site (IRES), ribosome shunting, and eIF4G enhancers. These mRNAs typically code for oncogenic, survival, and cell stress molecules. Among those mRNAs translationally increased by equol was the oncogene and eIF4G enhancer, c-Myc. Here we report that siRNA-mediated knockdown of c-Myc abrogates the increase in cancer cell viability and mammosphere formation by equol, and results in a significant down-regulation of eIF4GI (the major eIF4G isoform), as well as reduces levels of some, but not all, proteins encoded by mRNAs that are translationally stimulated by equol treatment. Knockdown of eIF4GI also markedly reduces an equol-mediated increase in IRES-dependent mRNA translation and the expression of specific oncogenic proteins. However, eIF4GI knockdown did not reciprocally affect c-Myc levels or cell viability. This study therefore implicates c-Myc as a potential regulator of the cancer-promoting effects of equol via up-regulation of eIF4GI and selective initiation of translation on mRNAs that utilize non-canonical initiation, including certain oncogenes.