Rho GTPases Rac1 Research Articles

Abstract 2185: Efficacy of Rac/Cdc42 inhibitors in multiple cancer models

Abstract The RhoGTPases Rac and Cdc42 are excellent therapeutic targets for inhibiting metastasis because these homologous proteins regulate numerous cellular functions that promote tumorigenesis, angiogenesis, invasion and therefore, metastasis. Also, Rac1, Cdc42, and their effector p21-activated kinase (PAK) are overexpressed in multiple cancer types, including breast, gastric, and pancreatic, and are associated with higher mortality rates. Furthermore, Rac and Cdc42 are activated through the signaling of oncogenic cell surface receptors such as EGFR and HER2, therefore hyperactivating their signaling pathways. Our laboratory has developed two small molecules targeting Rac and Cdc42, Ehop-016 and MBQ-167, that inhibit Rac1 and Cdc42 with IC50s in the low micro- and nano-molar concentrations, respectively (Montalvo-Ortiz et al. 2012, J Biol Chem; Humphries-Bickley et al. 2017, Mol Cancer Therap), making them the most potent Rac/Cdc42 inhibitors developed to date. We hypothesize that targeting Rac1, 3, and Cdc42 is a rational strategy to inhibit aggressive TNBC, pancreatic and gastric cancer. To test this hypothesis, we treated the TNBC cell lines MDA-MB-231, MDA-MB-468 and 4T1 (murine breast cancer), gastric cancer cells NCI-N87, and pancreatic cancer cells MIA-PaCa-2 with 4uM and 500nM of Ehop-016 and MBQ-167 respectively, and measured cell viability. Both TNBC cell lines and the pancreatic cancer cell line exhibited a drastic reduction in cell viability (&gt60%) after 48hrs of treatment Ehop-016 or MBQ-167. Additionally, experimental and spontaneous metastasis assays were performed with TNBC and gastric cancer xenografts in immunocompromised and immunocompetent mice. Treatment with 1 mg/kg BW MBQ-167 3X week resulted in reduced tumor growth by 60-90% in TNBC and gastric cancer models, and metastasis inhibition by ~90% in the MDA-MB-231 model. In the syngeneic experimental metastasis study with 4T1, both Ehop-016 and MBQ-167 reduced lung metastasis in comparison to the vehicle treated group. Moreover, these compounds were not toxic to both immunocompromised and immunocompetent mice, as observed by no significant changes in weight and liver enzymes. Therefore, both Ehop-016 and MBQ-167 are promising targeted therapeutics against cancers that overexpress Rac, Cdc42, and PAK. Citation Format: Jean F. Ruiz Calderon, Linette Castillo-Pichardo, Irmaris Lopez-Lopez, Suranganie Dharmawardhane. Efficacy of Rac/Cdc42 inhibitors in multiple cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2185.

Engineering proteins for allosteric control by light or ligands.

Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light-oxygen-voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.

Non-canonical WNT-signaling controls differentiation of lymphatics and extension lymphangiogenesis via RAC and JNK signaling

Development of lymphatics takes place during embryogenesis, wound healing, inflammation, and cancer. We previously showed that Wnt5a is an essential regulator of lymphatic development in the dermis of mice, however, the mechanisms of action remained unclear. Here, whole-mount immunostaining shows that embryonic day (ED) 18.5 Wnt5a-null mice possess non-functional, cyst-like and often blood-filled lymphatics, in contrast to slender, interconnected lymphatic networks of Wnt5a+/− and wild-type (wt) mice. We then compared lymphatic endothelial cell (LEC) proliferation during ED 12.5, 14.5, 16.5 and 18.5 between Wnt5a−/−, Wnt5a+/− and wt-mice. We did not observe any differences, clearly showing that Wnt5a acts independently of proliferation. Transmission electron microscopy revealed multiple defects of LECs in Wnt5a-null mice, such as malformed inter-endothelial junctions, ruffled cell membrane, intra-luminal bulging of nuclei and cytoplasmic processes. Application of WNT5A protein to ex vivo cultures of dorsal thoracic dermis from ED 15.5 Wnt5a-null mice induced flow-independent development of slender, elongated lymphatic networks after 2 days, in contrast to controls showing an immature lymphatic plexus. Reversely, the application of the WNT-secretion inhibitor LGK974 on ED 15.5 wt-mouse dermis significantly prevented lymphatic network elongation. Correspondingly, tube formation assays with human dermal LECs in vitro revealed increased tube length after WNT5A application. To study the intracellular signaling of WNT5A we used LEC scratch assays. Thereby, inhibition of autocrine WNTs suppressed horizontal migration, whereas application of WNT5A to inhibitor-treated LECs promoted migration. Inhibition of the RHO-GTPase RAC, or the c-Jun N-terminal kinase JNK significantly reduced migration, whereas inhibitors of the protein kinase ROCK did not. WNT5A induced transient phosphorylation of JNK in LECs, which could be inhibited by RAC- and JNK-inhibitors. Our data show that WNT5A induces formation of elongated lymphatic networks through proliferation-independent WNT-signaling via RAC and JNK. Non-canonical WNT-signaling is a major mechanism of extension lymphangiogenesis, and also controls differentiation of lymphatics.

Abstract P6-21-14: Targeting Rac/Cdc42 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer

Abstract Breast cancer metastasis, when cancer cells move and establish tumors in distant organs, confounds treatment options. Therefore, there is an unmet need for targeted therapeutics to address metastasis, especially in the intractable HER2 positive (+) breast cancer type. HER2 (+) breast cancer is treated with the monoclonal HER2 antibody trastuzumab and the HER2/EGFR targeting small molecule lapatinib. However, ˜20% of early stage breast cancer patients and ˜70% of patients with metastatic disease are resistant to trastuzumab. Trastuzumab-resistant breast cancers circumvent HER2 inhibition via bypass signaling, which includes the PI3-K/Akt/mTOR pathway. HER2/EGFR and PI-3K/Akt signaling activate the Rho GTPases Rac and Cdc42, and overexpression of Rac or its downstream effector p21-activated kinase (PAK) significantly diminishes response to anti HER2 therapy and patient survival. Therefore, targeting Rac and Cdc42, pivotal regulators of cancer cell migration/invasion, and thus, metastasis, is a viable option to overcome HER2/EGFR therapy resistance. First, we designed and developed the Rac inhibitor Ehop-016 (US patents 8,884,006 B2, 9,278,956B1), which inhibits Rac activation with an IC50 of 1 μM and reduces breast cancer growth and metastasis in mouse models of trastuzumab resistant HER2 (+) breast cancer. A more potent dual Rac and Cdc42 inhibitor, MBQ-167 (US Patent 9,981,980 B2), inhibits Rac and Cdc42 activation with IC50s of 103 nM and 78 nM, respectively. In vivo, MBQ-167 inhibits trastuzumab resistant HER2 (+) mammary tumor growth and metastasis by ˜90-100% (Humphries-Bickley et al. 2017, Mol Cancer Therap). Therefore, Rac/Cdc42 inhibition blocks cell proliferation, cell cycle progression, migration, and induces apoptosis to ultimately impede tumor growth and metastasis in trastuzumab resistant HER2 (+) breast cancer models. To further investigate the role of Rac/Cdc42 inhibitors in overcoming HER2 therapy resistance, we developed a lapatinib resistant variant of the SKBR3 HER2 (+) breast cancer cell line and found that Rac was overexpressed and over activated in the therapy resistant variant compared to the parental cells. Accordingly, the Rac/Cdc42 inhibitors overcame lapatinib resistance by decreasing cell viability and inducing apoptosis in parental and SKBR3 therapy resistant variants. Pharmacokinetic analysis in mouse plasma demonstrated that the bioavailability of EHop-016 and MBQ-167 is ˜30% with a half-life of 3-4 h. Therefore, to increase the bioavailability of Rac/Cdc42 inhibitors and to facilitate sensitization of trastuzumab therapy, we designed and developed novel nanoliposomal formulations containing Rac/Cdc42 inhibitors with trastuzumab on the outer surface to target HER2 (+) breast cancer cells. This data demonstrates the utility of developing Rac/Cdc42 inhibitors as mono or combined therapy with current targeted therapeutics for HER2 (+) breast cancer. Citation Format: Rivera-Robles MJ, Medina-Vlazquez JI, Borrero-Garcia LD, Velazquez-Vega LE, Maldonado MdM, Ruiz-Calderon J, Castillo-Pichardo L, Vivas-Mejia P, Dharmawardhane S. Targeting Rac/Cdc42 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-21-14.

Cullin-3/KCTD10 E3 complex is essential for Rac1 activation through RhoB degradation in human epidermal growth factor receptor 2-positive breast cancer cells.

Rho GTPase Rac1 is a central regulator of F‐actin organization and signal transduction to control plasma membrane dynamics and cell proliferation. Dysregulated Rac1 activity is often observed in various cancers including breast cancer and is suggested to be critical for malignancy. Here, we showed that the ubiquitin E3 ligase complex Cullin‐3 (CUL3)/KCTD10 is essential for epidermal growth factor (EGF)‐induced/human epidermal growth factor receptor 2 (HER2)‐dependent Rac1 activation in HER2‐positive breast cancer cells. EGF‐induced dorsal membrane ruffle formation and cell proliferation that depends on both Rac1 and HER2 were suppressed in CUL3‐ or KCTD10‐depleted cells. Mechanistically, CUL3/KCTD10 ubiquitinated RhoB for degradation, another Rho GTPase that inhibits Rac1 activation at the plasma membrane by suppressing endosome‐to‐plasma membrane traffic of Rac1. In HER2‐positive breast cancers, high expression of Rac1 mRNA significantly correlated with poor prognosis of the patients. This study shows that this novel molecular axis (CUL3/KCTD10/RhoB) positively regulates the activity of Rac1 in HER2‐positive breast cancers, and our findings may lead to new treatment options for HER2‐ and Rac1‐positive breast cancers.

Visualization of translocons in Yersinia type III protein secretion machines during host cell infection.

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery—also named injectisome—assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.

Activated Rho GTPases in Cancer-The Beginning of a New Paradigm.

Involvement of Rho GTPases in cancer has been a matter of debate since the identification of the first members of this branch of the Ras superfamily of small GTPases. The Rho GTPases were ascribed important roles in the cell, although these were restricted to regulation of cytoskeletal dynamics, cell morphogenesis, and cell locomotion, with initially no clear indications of direct involvement in cancer progression. This paradigm has been challenged by numerous observations that Rho-regulated pathways are often dysregulated in cancers. More recently, identification of point mutants in the Rho GTPases Rac1, RhoA, and Cdc42 in human tumors has finally given rise to a new paradigm, and we can now state with confidence that Rho GTPases serve as oncogenes in several human cancers. This article provides an exposé of current knowledge of the roles of activated Rho GTPases in cancers.

Loss of ARHGEF6 Causes Hair Cell Stereocilia Deficits and Hearing Loss in Mice.

ARHGEF6 belongs to the family of guanine nucleotide exchange factors (GEFs) for Rho GTPases, and it specifically activates Rho GTPases CDC42 and RAC1. Arhgef6 is the X-linked intellectual disability gene also known as XLID46, and clinical features of patients carrying Arhgef6 mutations include intellectual disability and, in some cases, sensorineural hearing loss. Rho GTPases act as molecular switches in many cellular processes. Their activities are regulated by binding or hydrolysis of GTP, which is facilitated by GEFs and GTPase-activating proteins, respectively. RAC1 and CDC42 have been shown to play important roles in hair cell (HC) stereocilia development. However, the role of ARHGEF6 in inner ear development and hearing function has not yet been investigated. Here, we found that ARHGEF6 is expressed in mouse cochlear HCs, including the HC stereocilia. We established Arhgef6 knockdown mice using the clustered regularly interspaced short palindromic repeat-associated Cas9 nuclease (CRISPR-Cas9) genome editing technique. We showed that ARHGEF6 was indispensable for the maintenance of outer hair cell (OHC) stereocilia, and loss of ARHGEF6 in mice caused HC stereocilia deficits that eventually led to progressive HC loss and hearing loss. However, the loss of ARHGEF6 did not affect the synapse density and did not affect the mechanoelectrical transduction currents in OHCs at postnatal day 3. At the molecular level, the levels of active CDC42 and RAC1 were dramatically decreased in the Arhgef6 knockdown mice, suggesting that ARHGEF6 regulates stereocilia maintenance through RAC1/CDC42.

NADPH-oxidase-derived ROS alterscell migration by modulating adhesions dynamics.

Cell migration requires the coordinated activation of structural and signalling molecules, such as the RhoGTPase Rac1. It is known that the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex assembly, which generates reactive oxygen species (ROS) at the cell membrane, also relies on Rac1 activation, indicating a possible effect of ROS during cell migration. In this study, we evaluated the effect of NADPH-oxidase-derived ROS on the migration process. Using time-lapse videos of CHO.K1 cells plated on fibronectin (2μg/ml) or collagen (5μg/cm2 ), we observed that depletion of ROS by N-acetyl-cysteine (NAC, 10mM), an unspecific antioxidant, or diphenyliodonium (DPI, 10μM), a NADPH-oxidase inhibitor, induced a ∼50% decrease in migration speed and severely impacted migration directionality. Then, we analysed the effects of NADPH oxidase on three migratory events: protrusion rate, adhesion process and signalling pathways related to cell migration. DPI induced an increase of ∼3 protrusion/cell, which were 2× faster but had a ∼50% retraction when compared with control. By pull-down assay, we observed no changes on Rac1 activation, indicating that ROS-mediated effects were related to downstream molecules, such as adhesion-related molecules. A reduction of the adhesion marker FAK-Y397 levels in cells treated with NAC and DPI was observed. In order to analyse adhesion dynamics, CHO.K1 cells transfected with paxillin-GFP analysed with total internal reflectance fluorescence (TIRF) indicated that DPI (5μM) induced larger adhesions when compared with control. These results indicate that the local generation of NADPH-oxidase-derived ROS can modulate cell migration due to changes on adhesion dynamics and signalling. This study highlights the physiological requirement of ROS for cell migration and the potential use of these molecules as targets to modulate the cell migration process at different diseases.

PARP-1-dependent RND1 transcription induced by topoisomerase I cleavage complexes confers cellular resistance to camptothecin

RHO GTPases regulate essential functions such as the organization of the actin cytoskeleton. The classic members cycle between an active GTP-bound and an inactive GDP-bound conformation whereas atypical members are predominantly GTP-bound. Besides their well-established role, the classic RHO GTPases RHOB and RAC1, are rapidly induced and/or activated by genotoxic stress and contribute to the DNA damage response. Here we used camptothecin, a selective topoisomerase I (TOP1) inhibitor that stabilizes TOP1 cleavage complexes (TOP1cc), to search for other potential early DNA damage-inducible RHO GTPase genes. We identified that an atypical RHO GTPase, RND1, is rapidly induced by camptothecin. RND1 induction is closely associated with the presence of TOP1cc induced by camptothecin or by DNA lesions that elevate TOP1cc levels such as UV and hydrogen peroxide. We further demonstrated that camptothecin increases RND1 gene transcription and mRNA stability. Camptothecin also increases poly(ADP-ribose) polymerase 1 (PARP-1) activity, whose inhibition reduces RND1 transcription. In addition, overexpression of RND1 increases PARP-1, suggesting a cross-talk between PARP-1 and RND1. Finally, RND1 protects cells against camptothecin-induced apoptosis, and hence favors cellular resistance to camptothecin. Together, these findings highlight RND1 as an atypical RHO GTPase early induced by TOP1cc, and show that the TOP1cc-PARP-1-RND1 pathway protects cells against apoptosis induced by camptothecin.

Actin cytoskeleton dynamics in stem cells from autistic individuals

Several lines of indirect evidence, such as mutations or dysregulated expression of genes related to cytoskeleton, have suggested that cytoskeletal dynamics, a process essential for axons and dendrites development, is compromised in autism spectrum disorders (ASD). However, no study has yet examined whether cytoskeleton dynamics is functionally altered in cells from ASD patients. Here we investigated the regulation of actin cytoskeleton dynamics in stem cells from human exfoliated deciduous teeth (SHEDs) of 13 ASD patients and 8 control individuals by inducing actin filament depolymerization and then measuing their reconstruction upon activation of the RhoGTPases Rac, Cdc42 or RhoA. We observed that stem cells from seven ASD individuals (53%) presented altered dymanics of filament reconstruction, including a patient recently studied by our group whose iPSC-derived neuronal cells show shorten and less arborized neurites. We also report potentially pathogenic genetic variants that might be related to the alterations in actin repolymerization dynamics observed in some patient-derived cells. Our results suggest that, at least for a subgroup of ASD patients, the dynamics of actin polymerization is impaired, which might be ultimately leading to neuronal abnormalities.

Abstract 4185: The novel Rac inhibitor HV-107 as a potential therapeutic for metastatic breast cancer

Abstract Breast cancer is the first cause of death in women globally. Metastatic breast cancer, which occurs when cancer cells migrate from the primary tumor and establish in distant organs, is estimated to affect more than a quarter of a million of women in the US. Regrettably, metastatic breast cancer still lacks effective treatment and remains the principal cause of breast cancer mortality. Therefore, it is imperative to develop new and more effective strategies to inhibit metastasis. A promising target for anti-metastatic cancer therapy is the Rho GTPase Rac because it plays a key role in the regulation of cellular adhesion, migration, proliferation and survival; processes which promote tumorigenesis and metastasis. Recently, our group developed Ehop-016, a small molecule that inhibits Rac in metastatic breast cancer cells with an IC50 of 1µM and reduces the activity of its downstream effector p21-activated kinase (PAK). Ehop-016 also inhibits tumor growth, angiogenesis, and metastasis in a mouse model of metastatic breast cancer. However, its relative bioavailability is moderate and should be further improved. Therefore, in an effort to find a compound with greater inhibitory capacity and bioavailability, we tested several Ehop-016 derivatives for their activity against Rac and their toxicity towards metastatic breast cancer cells. Using Rac pulldown assays we show that HV-107 at 250nM inhibits Rac activation by 55% in MDA-MB-231 and MDA-MB-435 metastatic breast cancer cells. The effects of HV-107 on cell viability were tested by MTT assays and our results indicate HV-107 at ≥500nM significantly inhibits metastatic breast cancer cell viability, while showing minimal toxicity towards non-cancerous mammary epithelial cells. We also measured caspase 3 activity, in order to evaluate apoptosis as a potential mechanism of HV-107-induced cell death. Our results show HV-107 at 1000nM causes a significant 1.5 fold increase in caspase 3 activity in MDA-MB-231 cells, indicating HV-107 affects cell viability by inducing apoptosis. Finally, we tested HV-107 in a mouse model of metastatic breast cancer and found a 25% reduction in tumor growth in mice treated with 5mg/kg BW HV-107. Taken together, our results suggest HV-107 is approximately four times more efficient than the parent compound Ehop-016 and has potential as anti-breast cancer metastasis therapeutic. This study was supported by awards from the Susan Komen for the Cure, NIH/NIMHHD U54MD008149, and the Puerto Rico Science and Technology Trust to SD; NIH/NCRR R25GM061838 to UPR MSC; NIH/NIMHHD RCMI 8G12MD007583RCMI, Title V PPOHA 031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education to UCC; and PRINBRE (NIH/NIGMS P20GM103475-13) Sub-Award to LCP. Citation Format: Grace E. Velez Crespo, Eliud Hernández, Cornelis Vlaar, Suranganie Dharmawardhane, Linette Castillo-Pichardo. The novel Rac inhibitor HV-107 as a potential therapeutic for metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4185.

Abstract 1905: New targeted therapeutics for gastric cancer

Abstract Gastric cancer is a health disparity with 2.3 times higher incidence rates in Hispanics than in Non-Hispanic Whites (NHWs). Very few studies have been conducted to characterize this health disparity in Hispanics; however, studies from East Asian patients, where gastric cancer is also a health disparity, have revealed that p21-activated kinases (PAKs 1,2 and 4) are biomarkers for gastric cancer. Even though inhibition of PAKs impedes gastric cancer progression, current PAK inhibitors have failed to successfully complete clinical trials. Thus, we hypothesize that the inhibition of upstream effectors of PAK, the Rho GTPases Rac and Cdc42 is a rational treatment for gastric cancers with high PAK expression. The purpose of this study is to correlate Rac/Cdc42 and PAK expression and activation with Hispanic gastric cancer malignancy, and to test the efficacy of our recently developed Rac/Cdc42 inhibitor MBQ-167 (Humphries-Bickley, et al., Mol Cancer Therap. 2017, 16:805-818). In a related Aim, to identify new targets and biomarkers for Hispanic gastric cancer, gastric cancer tissues and normal gastric mucosa from biopsies and/or surgery were collected from Puerto Rican patients for expression profiling with customized qRT-PCR arrays for mRNA and Agilent arrays for non-coding RNAs (ncRNAs), and western blotting. Western blotting of gastric cancer cell lines revealed that NCI-N87 metastatic gastric cancer cells express more PAK isoforms and phospho (active)-PAK compared to the AGS non-metastatic gastric cancer cells; accordingly, MBQ-167 reduces the viability of NCI-N87 cells without affecting AGS cells. Therefore, to test the efficacy of Rac/Cdc42 inhibition in gastric cancer, immunocompromised mice with subcutaneous tumors from GFP-NCI-N87 cells were treated 3X week with vehicle or 1mg/kg BW of MBQ-167. Tumor growth calculated by image J analysis of fluorescent digital images. A drastic reduction in NCI-N87 tumor growth was observed in the MBQ-167 treated mice with a 61% inhibition in tumor size compared to the controls, and a tumor growth delay of >90% after the second doubling time. Gene expression profiling revealed that 13 genes were overexpressed (>1.5 fold change) in Puerto Rican gastric cancer tissue in comparison to the normal gastric mucosa, including Rac1, PAK1, STAT3, KDR (VEGFR2), CXCR4, IGF1, VEGFA, and IL-6, all of which are associated with Rac/Cdc42/Pak signaling. Differential profiling using a custom-made Agilent array for ncRNAs demonstrated upregulation of a number of pro-cancer microRNAs (miRNAs) such as miR-106b and downregulation of tumor suppressors such as let-7a and miR-145, as well as upregulation of the long ncRNA CCAT2, which we have shown to be important for colon cancer malignancy. Taken together, our data show that Hispanic gastric cancers have known and novel biomarkers, and MBQ-167 is a viable targeted therapeutic option for gastric cancers with elevated Rac/Cdc42/PAK signaling. Citation Format: Jean F. Ruiz Calderon, Marcia Cruz-Correa, George A. Calin, Linette Castillo-Pichardo, Suranganie Dharmawardhane. New targeted therapeutics for gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1905.

Abstract 4923: Pharmacokinetics of the metastatic cancer inhibitor MBQ-167 in mice

Abstract Metastasis is the principal cause of death for most cancer patients; however, effective treatment options are limited. The Rho GTPases Rac and Cdc42 are critical regulators of cancer cell migration and invasion; processes required for metastatic progression. Hence, targeting of these pivotal regulators is a rational approach for novel metastasis focused therapy design. We previously characterized the small molecule MBQ-167 as a novel Rac/Cdc42 inhibitor that inhibits mammary tumor growth and metastasis in immunocompromised mice by ~90%, which is ten times more potent than other currently available Rac inhibitors (Humphries-Bickley, et al., 2017). However, further studies are needed to determine the pharmacokinetic (PK) parameters of MBQ-167 in rodents to understand the mechanisms of drug distribution and elimination of this compound. The purpose of this study was to determine the pharmacokinetics and bioavailability of MBQ-167 in a single dose input scheme (10 mg/kg BW) following intraperitoneal (IP) and oral gavage (PO) administration. The drug was administered to BALB/c mice (4 mice/group) and plasma was collected at 0.5, 1, 3, 6, 9, and 12 hours after IP or PO administration. We developed and validated a bioanalytical method using supercritical fluid chromatography (SFC) coupled with electrospray ionization tandem mass spectrometry (MS/MS) for the detection of MBQ-167 in plasma. SFC-MS/MS was selected because it uses supercritical carbon dioxide (SCO2) as the mobile phase and offers advantages such as high sensitivity, increased resolution, and rapid analysis times. Pharmacokinetic parameters were obtained by mono-compartmental and bi-compartmental analysis for IP and PO dosing respectively using WinNolin® software, Version 7.0. Preliminary pharmacokinetic analysis revealed that the area under the curve (AUC0-∞) was 749 ng·hr/mL and 255 ng·hr/mL for IP and PO dosing respectively. The elimination half-life (t1/2) was 2.4 hours for IP dosing and the mean residence time (MRT) was 3.4 hours and 2.3 hours for the IP and PO dosing respectively. The maximum plasma drug concentration (Cmax) was 220 ng/mL after IP administration, compared to 127 ng/mL following PO administration. Furthermore, the time-to-peak for both administration routes was 30 minutes. The relative bioavailability of MBQ-167 after oral gavage administration was 34%. This study presents the first analysis of the pharmacokinetics of the Rac/Cdc42 inhibitor MBQ-167 in mice and it supports the continued development of this drug as a potential anti-cancer therapeutic. Citation Format: Maria del Mar Maldonado, Linette Castillo-Pichardo, Joseph Bloom, Jorge Duconge, Jose F. Rodriguez-Orengo, Eliud Hernandez-O'Farrill, Cornelis Vlaar, Suranganie Dharmawardhane. Pharmacokinetics of the metastatic cancer inhibitor MBQ-167 in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4923.

Abstract 4505: Silencing connexin 43 induces cell cycle entry and invasion in non-neoplastic mammary epithelial cells

Abstract Studies demonstrated loss of connexin 43 (Cx43) expression in breast cancer cells in-vitro and in-vivo. Our previous studies have shown that silencing Cx43 in HMT-3522 S1 cells, non-neoplastic human mammary epithelial cells (S1 cells), altered apical polarity and mitotic spindle orientation under 3-dimensional (3-D) culture conditions. The purpose of this study is to investigate the effects of Cx43 loss on proliferation and invasion of non-neoplastic breast epithelium. Cx43 shRNA-transfected and control S1 cells were cultured under 2-D and 3-D conditions. Cx43-knockdown S1 cells exhibited enhanced proliferation both in 2-D and 3-D throughout the culture period, as shown by the increased cell counts and acinar size, respectively. In addition, cell cycle entry was enhanced, as indicated by the increased percentages of cells in S and G2/M phases and the reduced percentage of cells in G0/G1 phase. Cx43-silenced S1 cells upregulated the expression of proliferation genes, c-Myc and cyclin D1, in 2-D and 3-D, but maintained β-catenin levels. Furthermore, immunoprecipitation studies showed that Cx43 associated with β-catenin and ZO-2 in 2-D and 3-D cultures of S1 cells, and co-localized with β-catenin in 3-D, suggesting assembly of gap junction complexes in S1 cells. The loss of Cx43 caused mis-localization of β-catenin in 3-D cultures of S1 cells, suggesting involvement of the Wnt/β-catenin pathway downstream of Cx43 signaling. β-Catenin primarily exhibited apico-lateral distribution in S1 cells and re-localized to basolateral membrane domains in Cx43 knockdown cells. Silencing Cx43 enhanced migration of S1 cells by 40%, as shown by time-lapse imaging. In addition, Cx43-silenced S1 cells exhibited loss of the characteristic spheroid morphology and acquired a migratory phenotype in acini. Matrigel-invasion and MMP-9 activity were also enhanced. Cx43-silenced S1 cells were capable of invading the matrigel by two-fold compared to control cells. This was concomitant with enhanced expression and activity levels of Rho GTPases (RhoA, Rac1 and Cdc42), suggesting that Cx43 regulates non-canonical Wnt signaling, as a mechanism to induce migration and invasion. We propose a role for Cx43 loss in triggering proliferation and invasion events in non-neoplastic breast epithelium downstream of disrupted Wnt signaling. Citation Format: Sabreen F. Fostok, Dana Bazzoun, Sophie A. Lelievre, Mirvat El-Sibai, Rabih S. Talhouk. Silencing connexin 43 induces cell cycle entry and invasion in non-neoplastic mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4505.

RhoD localization and function is dependent on its GTP/GDP-bound state and unique N-terminal motif

The atypical Rho GTPase RhoD has previously been shown to have a major impact on the organization and function of the actin filament system. However, when first discovered, RhoD was found to regulate endosome trafficking and dynamics and we therefore sought to investigate this regulation in more detail. We found that exogenously expressed RhoD in human fibroblasts localized to vesicles and the plasma membrane and that the active GTP-bound conformation was required for the plasma membrane localization but not for vesicle localization. In contrast to the GTPase deficient atypical Rho GTPases, which have a stalled GTPase activity, RhoD has an elevated intrinsic GDP/GTP exchange activity, rendering the protein constitutively active. Importantly, RhoD can still hydrolyze GTP and we found that an intact GTPase activity was required for efficient fusion of RhoD-positive vesicles. RhoD has a unique N-terminal extension of 14 amino acid residues, which is not present in the classical Rho GTPases RhoA, Cdc42 and Rac1. Deletion of this N-terminal motif often lead to clustering of RhoD positive vesicles, which were found accumulated at the peripheral membrane border. In addition, the number of vesicles per cell was increased manifold, suggesting that the N-terminal motif has an important regulatory role in vesicle dynamics.

Abstract A44: Establishment of patient-derived Hispanic gastric cancer xenografts to test targeted therapeutics

Abstract Gastric cancer is a health disparity with 2.3 times higher incidence rates in Hispanics than in non-Hispanic Whites (NHWs). Very few studies have been conducted to characterize this health disparity in Hispanics; however, studies from East Asian patients, in whom gastric cancer is also a health disparity, have revealed that p21-activated kinases (PAKs 1,2 and 4), which are known to regulate cancer metastasis, are biomarkers for gastric cancer. Even though inhibition of PAKs impedes gastric cancer progression, current PAK inhibitors have failed to successfully complete clinical trials. Thus, we hypothesize that the inhibition of upstream effectors of PAK, the Rho GTPases Rac and Cdc42, is a rational treatment for gastric cancers with high PAK expression. The purpose of this study is to identify the Rac/Cdc42/PAK pathway as novel biomarkers for Hispanic gastric cancer and to test targeted therapeutics in patient-derived gastric cancer xenografts (PDGCXs). To achieve this aim, gastric cancer tissues and normal gastric mucosa from biopsies and/or surgery are being collected for expression profiling with customized qRT-PCR arrays for mRNA and Agilent arrays for noncoding RNAs (ncRNAs), and Western blotting. Using immunodeficient mice, xenografts have been established from the NCI-N87 gastric cancer cell line (from ATCC) and a tumor from a 58-year-old Hispanic (Puerto Rican) female (San Juan Oncology Hospital). As demonstrated by Western blotting, compared to the AGS nonmetastatic gastric cancer cell line, the NCI-N87 metastatic gastric cancer cells express more PAK isoforms and phospho (active)-PAK. We have also shown that our recently developed Rac/Cdc42 inhibitor MBQ-167 reduces the viability of the NCI-N87 cell line (Humphries-Bickley et al., Mol Cancer Therap 2017;16:805-18). Therefore, we are currently testing the effect of MBQ-167 in SCID mice with NCI-N87 xenografts. Gene expression profiling of the gastric cancer tissue from the Hispanic patient revealed that 13 genes were overexpressed (>1.5-fold change) in gastric cancer tissue in comparison to the normal gastric mucosa, including Rac1, PAK1, STAT3, KDR (VEGFR2), CXCR4, IGF1, VEGFA, and IL-6, all of which are associated with Rac/Cdc42/Pak signaling. Differential profiling using a custom-made Agilent array for ncRNAs demonstrated upregulation of a number of procancer microRNAs (miRNAs) such as miR-106b and downregulation of tumor suppressors such as let-7a and miR-145, as well as upregulation of the long ncRNA CCAT2, which we have shown to be important for colon cancer malignancy. Overall, our data show that a number of unique and established biomarkers are present in Hispanic gastric cancer, including elevated Rac/Cdc42/PAK signaling. Therefore, treatment with drugs targeting the Rac/Cdc42/PAK pathway represents a novel therapeutic option for such gastric cancers. In conclusion, modeling gastric cancer in PDGCXs is expected to enable the testing and design of targeted and individualized treatments for Puerto Rican patients. Citation Format: Jean F. Ruiz-Calderon, Marcia Cruz-Correa, George A. Calin, Suranganie Dharmawardhane. Establishment of patient-derived Hispanic gastric cancer xenografts to test targeted therapeutics [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A44.

RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1.

RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS.

Novel Rac Inhibitors as Targeted Therapeutics for Metastatic Breast Cancer

Metastatic disease is the primary cause of breast cancer mortality, due to the lack of effective therapy. The Rho GTPase Rac is integral for the promotion of cancer cell migration/invasion, proliferation, and survival. Since metastatic breast cancers often overexpress or exhibit high Rac activity, inhibition of Rac is a viable strategy against metastatic cancer. Recently, we characterized EHop‐016, a small molecule that inhibits Rac activity of metastatic breast cancer cells with an IC50 of 1 μM. EHop‐016, which is 10–100 times more active than previously available Rac inhibitors, inhibits the activity of the Rac downstream effector p21 activated kinase (PAK), lamellipodia extension, and cell migration of metastatic breast cancer cells. Moreover, EHop‐016 at ≥ 25 mg/kg Body Weight (BW) significantly reduced tumor growth, metastasis, and angiogenesis in a mouse model. However, our recent pharmacokinetic study of EHop‐016 in a mouse model demonstrated that its bioavailability should to be improved for further pharmacological development. Our hypothesis is that improvement of the EHop‐016 structure will provide probes with increased potency against Rac and improved bioavailability. Herein we have tested several Ehop‐016 derivatives for their effects on breast cancer cell viability, cell migration, and Rac activation. Using MTT assays we found that the Ehop‐016 derivatives, HV‐107 and HV‐118, significantly inhibit the viability of metastatic breast cancer cell lines MDAMB‐231 and MDA‐MB‐435; while presenting minimal toxicity towards non‐cancerous mammary epithelial cells. The effects of HV‐107 and HV‐118 on the inhibition of Rac activation were tested by ELISA‐based Rac activity assays and pulldown assays. Results show that at 250 nM, HV‐107 inhibits Rac activation by 55%, whereas HV‐118 has a similar effect at 100 nM in MDA‐MB‐231 and MDA‐MB‐435 cells. Moreover, using transwell assays we studied the effects of both Rac inhibitors in metastatic breast cancer cell migration and observed a significant reduction. Finally, we tested HV‐107 in a mouse model of metastatic breast cancer and found a 25% reduction in tumor growth in mice treated with 5 mg/kg BW HV‐107. Taken together, our findings suggest HV‐107 and HV‐118 as promising Ehop‐016 derivatives with potential as anti‐metastatic agents, that should be further characterized.Support or Funding InformationThis study was supported by awards from the Susan Komen for the Cure, NIH/NIMHHD U54MD008149, and the Puerto Rico Science and Technology Trust to SD; NIH/NCRR R25GM061838 to UPR MSC; NIH/NIMHHD RCMI 8G12MD007583RCMI, Title V PPOHA 031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education to UCC; and PRINBRE (NIH/NIGMS P20GM103475‐13) Sub‐Award to LCP.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

N‐cadherin Signaling via RhoGEF Trio Stabilizes VE‐cadherin Junctions and Regulates Vascular Permeability

Capillary endothelial cells express Vascular Endothelial (VE)‐ and Neural (N)‐cadherin, with overlapping functions. VE‐cadherin forms homotypic adhesion between endothelial cells whereas N‐cadherin forms heterotypic adhesion with the surrounding pericytes in capillary endothelia. Endothelial specific deletions of Cdh2 (N‐cadherin) or Cdh5 (VE‐cadherin) in mice demonstrated poorly formed leaky capillaries and in utero death at E9.5 due to defective angiogenesis. These findings raise the question of whether N‐ and VE‐cadherin function independently or whether N‐cadherin activated signaling regulates the assembly of VE‐cadherin and thereby the formation of adherens junctions. We investigated the role of N‐cadherin in the formation of VE‐cadherin junctions using mouse genetic models and identifying N‐cadherin signaling pathways in endothelial monolayers. We show that N‐cadherin functions by interacting with the RhoGEF Trio to activate the RhoGTPases Rac1 and RhoA in nascent adherens junctions, inducing the recruitment of VE‐cadherin. This N‐cadherin activated signaling pathway is essential for maximal VE‐cadherin assembly and the formation of the endothelial junctional barrier.Support or Funding InformationSupported by NIH grant R01 HL103922 to Y.A.K.; AHA AWARD 16PRE27260230 to K.K.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.