guanosine-triphosphate and Breast-Neoplasms

One in eight women in the United States will be diagnosed with invasive breast cancer in her lifetime. Advances in therapeutic strategies, diagnosis, and improved awareness have resulted in a significant reduction in breast cancer related mortality. However, there is a continued need for more effective and less toxic drugs for both the prevention and the treatment of breast cancer in order to see a continued decline in the morbidity and mortality associated with this disease. Recent studies suggest that the cGMP signaling pathway may be aberrantly regulated in breast cancer. As such, this pathway may serve as a source of novel targets for future breast cancer drug discovery efforts. This review provides an overview of cGMP signaling in normal physiology and in breast cancer as well as current strategies being investigated for targeting this pathway in breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cyclic GMP; Female; Guanosine Triphosphate; Guanylate Cyclase; Humans; Models, Biological; Signal Transduction

Goserelin (GOS) therapy in an adjuvant setting for estrogen receptor(ER)-positive premenopausal patients with breast cancer was assessed in a randomised comparative study.. ER positive premenopausal patients with n + or n 0 and T > or = 3 cm received tamoxifen (TAM) 20 mg/day, GOS 3.6 mg/4 weeks or GOS + TAM for 2 years, and the clinical efficacy and safety of these regimens were assessed.. In the data analysis of total 207 patients, hazard ratios of disease free survival (DFS) and overall survival (OS) in the GOS group compared to the TAM group were 0.87 and 2.10,respectively. The incidence of adverse drug reactions was similar (42-55%) in all three groups. Since the number of patients in this study did not reach the target number, the efficacy could not be assessed from a statistical aspect. Therefore,meta-analysis with similar foreign studies(ZIPP) was implemented. The results of meta-analysis showed that the hazard ratios of DFS and OS in the GOS group compared to the non-GOS group were 0.83 and 0.85, respectively.. Although the analysis of 207 patients did not show any statistically significant difference between each of the treatment groups, the results of meta-analysis showed a significant prolongation of DFS in the GOS group. Also high tolerability of GOS was suggested. From these results, GOS was considered highly useful in adjuvant therapy for ER-positive premenopausal patients with breast cancer.

Topics: Adult; Antineoplastic Agents, Hormonal; Breast Neoplasms; Chemotherapy, Adjuvant; Drug Administration Schedule; Goserelin; Guanosine Triphosphate; Hot Flashes; Humans; Middle Aged; Premenopause; Proportional Hazards Models; Quality of Life; Receptors, Estrogen; Survival Rate; Tamoxifen

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death. Drug therapy for breast cancer is currently selected based on the subtype classification; however, many anticancer drugs are highly cytotoxic. Since intracellular levels of GTP are elevated in many cancer cells that undergo a specific cell proliferation cycle, GTP has potential as a target for cancer therapy. The present study focused on nucleosides and nucleotides and examined intracellular GTP-dependent changes in cell proliferation rates in normal (MCF-12A) and cancer (MCF-7) breast cell lines. Decreased cell proliferation due to a reduction in intracellular GTP levels by mycophenolic acid (MPA), an inosine monophosphate dehydrogenase inhibitor, was observed in both cell lines. The inhibitory effects of MPA on cell proliferation were suppressed when it was applied in combination with Guanosine (Guo), a substrate for GTP salvage synthesis, while the single exposure to Guo suppressed the proliferation of MCF-7 cells only. Although the underlying mechanisms remain unclear, since the inhibitory effects of Guo on cell proliferation did not correlate with GTP or ATP intracellular levels or the GTP/ATP ratio, there may be another cause besides GTP metabolism. Guo inhibited the proliferation of MCF-7, a human breast cancer cell line, but not MCF-12A, a human normal breast cell line. Further studies are needed to investigate the potential of applying Guo as a target for the development of a novel cancer treatment system.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Breast Neoplasms; Cell Proliferation; Female; Guanosine; Guanosine Triphosphate; Humans; MCF-7 Cells; Mycophenolic Acid

RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis. SIMPLE SUMMARY: Ran is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Gene Expression Regulation, Neoplastic; Guanosine Triphosphate; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; ran GTP-Binding Protein; Triple Negative Breast Neoplasms

Early metastasis is a key factor contributing to poor breast cancer (BC) prognosis. Circulating tumor cells (CTCs) are regarded as the precursor cells of metastasis, which are ultimately responsible for the main cause of death in BC. However, to date molecular mechanisms underlying CTC formation in BC have been insufficiently defined.. RNA-seq was carried out in primary tissues from early-stage BC patients (with CTCs≥5 and CTCs = 0, respectively) and the validation study was conducted in untreated 80 BC patients. Multiple in vitro and in vivo models were used in functional studies. Luciferase reporter, ChIP-seq, CUT&Tag-seq, and GST-pulldown, etc. were utilized in mechanistic studies. CTCs were counted by the CanPatrol™ CTC classification system or LiquidBiospy™ microfluidic chips. ERK1/2 inhibitor SCH772984 was applied to in vivo treatment.. Highly expressed FOXD1 of primary BC tissues was observed to be significantly associated with increased CTCs in BC patients, particularly in early BC patients. Overexpressing FOXD1 enhanced the migration capability of BC cells, CTC formation and BC metastasis, via facilitating epithelial-mesenchymal transition of tumor cells. Mechanistically, FOXD1 was discovered to induce RalA expression by directly bound to RalA promotor. Then, RalA formed a complex with ANXA2 and Src, promoting the interaction between ANXA2 and Src, thus increasing the phosphorylation (Tyr23) of ANXA2. Inhibiting RalA-GTP form attenuated the interaction between ANXA2 and Src. This cascade culminated in the activation of ERK1/2 signal that enhanced metastatic ability of BC cells. In addition, in vivo treatment with SCH772984, a specific inhibitor of ERK1/2, was used to dramatically inhibit the CTC formation and BC metastasis.. Here, we report a FOXD1-dependent RalA-ANXA2-Src complex that promotes CTC formation via activating ERK1/2 signal in BC. FOXD1 may serve as a prognostic factor in evaluation of BC metastasis risks. This signaling cascade is druggable and effective for overcoming CTC formation from the early stages of BC.

Topics: Annexin A2; Biomarkers, Tumor; Breast Neoplasms; Epithelial-Mesenchymal Transition; Female; Forkhead Transcription Factors; Guanosine Triphosphate; Humans; Neoplastic Cells, Circulating; Proto-Oncogene Proteins pp60(c-src); ral GTP-Binding Proteins

The small GTPase Cdc42 is an integral component of the cytoskeleton, and its dysregulation leads to pathophysiological conditions, such as cancer. Binding of Cdc42 to the scaffold protein IQGAP1 stabilizes Cdc42 in its active form. The interaction between Cdc42 and IQGAP1 enhances migration and invasion of cancer cells. Disrupting this association could impair neoplastic progression and metastasis; however, no effective means to achieve this has been described. Here, we screened 78,500 compounds using a homogeneous time resolved fluorescence-based assay to identify small molecules that disrupt the binding of Cdc42 to IQGAP1. From the combined results of the validation assay and counter-screens, we selected 44 potent compounds for cell-based experiments. Immunoprecipitation and cell viability analysis rendered four lead compounds, namely NCGC00131308, NCGC00098561, MLS000332963 and NCGC00138812, three of which inhibited proliferation and migration of breast carcinoma cells. Microscale thermophoresis revealed that two compounds bind directly to Cdc42. One compound reduced the amount of active Cdc42 in cells and effectively impaired filopodia formation. Docking analysis provided plausible models of the compounds binding to the hydrophobic pocket adjacent to the GTP binding site of Cdc42. In conclusion, we identified small molecules that inhibit binding between Cdc42 and IQGAP1, which could potentially yield chemotherapeutic agents.

Topics: Breast Neoplasms; cdc42 GTP-Binding Protein; Female; Guanosine Triphosphate; Humans; ras GTPase-Activating Proteins; Signal Transduction

As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 μg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.

Topics: Breast Neoplasms; Cell Transformation, Neoplastic; Female; Guanosine Triphosphate; Humans; rhoA GTP-Binding Protein; rhoC GTP-Binding Protein

RhoA stimulates cell contractility by recruiting downstream effectors to the cortical plasma membrane. We now show that direct binding by anillin is required for effective signaling: this antagonizes the otherwise labile membrane association of GTP-RhoA to promote effector recruitment. However, since its binding to RhoA blocks access by other effectors, we demonstrate that anillin must also concentrate membrane phosphoinositide-4,5-P

Topics: Breast Neoplasms; Cell Movement; Contractile Proteins; Cytokinesis; Female; Guanosine Triphosphate; Humans; Kinetics; MCF-7 Cells; Phosphatidylinositol 4,5-Diphosphate; rhoA GTP-Binding Protein; Signal Transduction

The small GTPase DiRas1 has tumor-suppressive activities, unlike the oncogenic properties more common to small GTPases such as K-Ras and RhoA. Although DiRas1 has been found to be a tumor suppressor in gliomas and esophageal squamous cell carcinomas, the mechanisms by which it inhibits malignant phenotypes have not been fully determined. In this study, we demonstrate that DiRas1 binds to SmgGDS, a protein that promotes the activation of several oncogenic GTPases. In silico docking studies predict that DiRas1 binds to SmgGDS in a manner similar to other small GTPases. SmgGDS is a guanine nucleotide exchange factor for RhoA, but we report here that SmgGDS does not mediate GDP/GTP exchange on DiRas1. Intriguingly, DiRas1 acts similarly to a dominant-negative small GTPase, binding to SmgGDS and inhibiting SmgGDS binding to other small GTPases, including K-Ras4B, RhoA, and Rap1A. DiRas1 is expressed in normal breast tissue, but its expression is decreased in most breast cancers, similar to its family member DiRas3 (ARHI). DiRas1 inhibits RhoA- and SmgGDS-mediated NF-κB transcriptional activity in HEK293T cells. We also report that DiRas1 suppresses basal NF-κB activation in breast cancer and glioblastoma cell lines. Taken together, our data support a model in which DiRas1 expression inhibits malignant features of cancers in part by nonproductively binding to SmgGDS and inhibiting the binding of other small GTPases to SmgGDS.

Topics: Amino Acid Sequence; Breast Neoplasms; Carcinoma, Ductal, Breast; GTP Phosphohydrolases; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; HEK293 Cells; Humans; MCF-7 Cells; Molecular Docking Simulation; NF-kappa B; Protein Binding; Protein Structure, Secondary; Proto-Oncogene Proteins p21(ras); rhoA GTP-Binding Protein; Tumor Suppressor Proteins

Most sporadic breast and ovarian cancers express low levels of the breast cancer susceptibility gene, BRCA1. The BRCA1 gene produces two transcripts, mRNAa and mRNAb. mRNAb, present in breast cancer but not in normal mammary epithelial cells, contains three upstream open reading frames (uORFs) in its 5'UTR and is translationally repressed. Comparable tandem uORFs are characteristically seen in mRNAs whose translational efficiency paradoxically increases when the overall translation rate is decreased due to phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α). Here we show fish oil derived eicosopanthenoic acid (EPA) that induces eIF2α phosphorylation translationally up-regulates the expression of BRCA1 in human breast cancer cells. We demonstrate further that a diet rich in EPA strongly induces expression of BRCA1 in human breast cancer xenografts.

Topics: 5' Untranslated Regions; Animals; BRCA1 Protein; Breast Neoplasms; Cell Line, Tumor; Eicosapentaenoic Acid; Eukaryotic Initiation Factor-2; Female; Fish Oils; Guanosine Triphosphate; Humans; MCF-7 Cells; Mice; Mice, Nude; Neoplasm Transplantation; Open Reading Frames; Phosphorylation; RNA Interference; RNA, Messenger; RNA, Transfer, Met

Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. Here we report that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduces NMG expression and substantially depletes nucleotide pools, which attenuates GTP-dependent protein activity and cell invasion. Addition of exogenous guanosine or GTP restores the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. In addition, mtp53 creates a dependency on the nucleoside salvage pathway enzyme deoxycytidine kinase for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53-harbouring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleoside salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53's control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.

Topics: Animals; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Deoxycytidine Kinase; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Guanosine Triphosphate; Humans; Immunoprecipitation; Kaplan-Meier Estimate; Mice; Mutation; Neoplasm Invasiveness; Neoplasm Transplantation; Nucleosides; Nucleotides; Prognosis; Promoter Regions, Genetic; Proportional Hazards Models; Tumor Stem Cell Assay; Tumor Suppressor Protein p53

For a solid tumor to grow, it must be able to support the compressive stress that is generated as it presses against the surrounding tissue. Although the literature suggests a role for the cytoskeleton in counteracting these stresses, there has been no systematic evaluation of which filaments are responsible or to what degree. Here, using a three-dimensional spheroid model, we show that cytoskeletal filaments do not actively support compressive loads in breast, ovarian, and prostate cancer. However, modulation of tonicity can induce alterations in spheroid size. We find that under compression, tumor cells actively efflux sodium to decrease their intracellular tonicity, and that this is reversible by blockade of sodium channel NHE1. Moreover, although polymerized actin does not actively support the compressive load, it is required for sodium efflux. Compression-induced cell death is increased by both sodium blockade and actin depolymerization, whereas increased actin polymerization offers protective effects and increases sodium efflux. Taken together, these results demonstrate that cancer cells modulate their tonicity to survive under compressive solid stress.

Topics: Actins; Adenocarcinoma; Azides; Biomechanical Phenomena; Breast Neoplasms; Cation Transport Proteins; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Cytoskeleton; Female; Guanosine Triphosphate; Humans; Models, Biological; Osmosis; Sodium; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Tissue Scaffolds

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis on G protein α subunits, restricting their activity downstream from G protein-coupled receptors. Here we identify Drosophila Double hit (Dhit) as a dual RGS regulator of Gαo. In addition to the conventional GTPase-activating action, Dhit possesses the guanine nucleotide dissociation inhibitor (GDI) activity, slowing the rate of GTP uptake by Gαo; both activities are mediated by the same RGS domain. These findings are recapitulated using homologous mammalian Gαo/i proteins and RGS19. Crystal structure and mutagenesis studies provide clues into the molecular mechanism for this unprecedented GDI activity. Physiologically, we confirm this activity in Drosophila asymmetric cell divisions and HEK293T cells. We show that the oncogenic Gαo mutant found in breast cancer escapes this GDI regulation. Our studies identify Dhit and its homologs as double-action regulators, inhibiting Gαo/i proteins both through suppression of their activation and acceleration of their inactivation through the single RGS domain.

Topics: 5' Untranslated Regions; Amino Acid Sequence; Animals; Breast Neoplasms; Crystallography, X-Ray; Drosophila melanogaster; Drosophila Proteins; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; GTP-Binding Protein alpha Subunits; Guanosine Triphosphate; HEK293 Cells; Humans; Hydrolysis; Molecular Sequence Data; Mutation; Open Reading Frames; Protein Structure, Tertiary; RGS Proteins; Signal Transduction; Time Factors

The expression of proinflammatory protein tissue transglutaminase 2 (TG2) is frequently upregulated in multiple cancer cell types. However, the exact role of TG2 in cancer cells is not well-understood. We recently initiated studies to determine the significance of TG2 in cancer cells and observed that sustained expression of TG2 resulted in epithelial-to-mesenchymal transition (EMT) and promoted cancer stem cell (CSC) traits in mammary epithelial cells. These results suggested that TG2 could serve as a promising therapeutic target for overcoming chemoresistance and inhibiting metastatic spread of cancer cells.. Using various mutant constructs, we analyzed the activity of TG2 that is essential for promoting the EMT-CSC phenotype.. Our results suggest that catalytically inactive TG2 (TG2-C277S) is as effective as wild-type TG2 (TG2-WT) in inducing the EMT-CSC in mammary epithelial cells. In contrast, overexpression of a GTP-binding-deficient mutant (TG2-R580A) was completely incompetent in this regard. Moreover, TG2-dependent activation of the proinflammatory transcription factor NF-κB is deemed essential for promoting the EMT-CSC phenotype in mammary epithelial cells.. Our results suggest that the transamidation activity of TG2 is not essential for promoting its oncogenic functions and provide a strong rationale for developing small-molecule inhibitors to block GTP-binding pockets of TG2. Such inhibitors may have great potential for inhibiting the TG2-regulated pathways, reversing drug resistance and inhibiting the metastasis of cancer cells.

Topics: Breast Neoplasms; Catalytic Domain; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Guanosine Triphosphate; Homeodomain Proteins; Humans; Mammary Glands, Human; Neoplasm Invasiveness; Neoplastic Stem Cells; NF-kappa B; Nuclear Proteins; Phenotype; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; Signal Transduction; Snail Family Transcription Factors; Spheroids, Cellular; Transcription Factors; Transglutaminases; Twist-Related Protein 1; Zinc Finger E-box-Binding Homeobox 1

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Shape; Female; Focal Adhesions; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Humans; Integrin beta1; Male; Microtubules; Neoplasm Invasiveness; Neoplasm Proteins; Prostatic Neoplasms; Pseudopodia; rac1 GTP-Binding Protein; Recombinant Fusion Proteins; Rho Guanine Nucleotide Exchange Factors; rhoB GTP-Binding Protein; RNA, Small Interfering

We performed a proteomics screen for Rho isoform-specific binding proteins to clarify the tumor-promoting effects of RhoA and C that contrast with the tumor-suppressive effects of RhoB. We found that the IQ-motif-containing GTPase-activating protein IQGAP1 interacts directly with GTP-bound, prenylated RhoA and RhoC, but not with RhoB. Co-immunoprecipitation of IQGAP1 with endogenous RhoA/C was enhanced when RhoA/C were activated by epidermal growth factor (EGF) or transfection of a constitutively active guanine nucleotide exchange factor (GEF). Overexpression of IQGAP1 increased GTP-loading of RhoA/C, while siRNA-mediated depletion of IQGAP1 prevented endogenous RhoA/C activation by growth factors. IQGAP1 knockdown also reduced the amount of GTP bound to GTPase-deficient RhoA/C mutants, suggesting that IQGAP enhances Rho activation by GEF(s) or stabilizes Rho-GTP. IQGAP1 depletion in MDA-MB-231 breast cancer cells blocked EGF- and RhoA-induced stimulation of DNA synthesis. Infecting cells with adenovirus encoding constitutively active RhoA(L63) and measuring absolute amounts of RhoA-GTP in infected cells demonstrated that the lack of RhoA(L63)-induced DNA synthesis in IQGAP1-depleted cells was not due to reduced GTP-bound RhoA. These data suggested that IQGAP1 functions downstream of RhoA. Overexpression of IQGAP1 in MDA-MB-231 cells increased DNA synthesis irrespective of siRNA-mediated RhoA knockdown. Breast cancer cell motility was increased by expressing a constitutively-active RhoC(V14) mutant or overexpressing IQGAP1. EGF- or RhoC-induced migration required IQGAP1, but IQGAP1-stimulated migration independently of RhoC, placing IQGAP1 downstream of RhoC. We conclude that IQGAP1 acts both upstream of RhoA/C, regulating their activation state, and downstream of RhoA/C, mediating their effects on breast cancer cell proliferation and migration, respectively.

Topics: Binding, Competitive; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Guanosine Triphosphate; HEK293 Cells; Humans; Immunoprecipitation; Protein Binding; Protein Prenylation; Proteomics; ras GTPase-Activating Proteins; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; rhoC GTP-Binding Protein; RNA Interference

The objective of this study was to determine whether geranylgeranyl diphosphate synthase inhibition, and therefore geranylgeranyl diphosphate depletion, interferes with breast cancer cell migration. Digeranyl bisphosphonate is a specific geranylgeranyl diphosphate synthase inhibitor. We demonstrate that digeranyl bisphosphonate depleted geranylgeranyl diphosphate and inhibited protein geranylgeranylation in MDA-MB-231 cells. Similar to GGTI-286, a GGTase I inhibitor, digeranyl bisphosphate significantly inhibited migration of MDA-MB-231 cells as measured by transwell assay. Similarly, digeranyl bisphosphonate reduced motility of MDA-MB-231 cells in a time-dependent manner as measured by large scale digital cell analysis system microscopy. Digeranyl bisphosphonate was mildly toxic and did not induce apoptosis. Treatment of MDA-MB-231 cells with digeranyl bisphosphonate decreased membrane while it increased cytosolic RhoA localization. In addition, digeranyl bisphosphonate increased RhoA GTP binding in MDA-MB-231 cells. The specificity of geranylgeranyl diphosphonate synthase inhibition by digeranyl bisphosphonate was confirmed by exogenous addition of geranylgeranyl diphosphate. Geranylgeranyl diphosphate addition prevented the effects of digeranyl bisphosphonate on migration, RhoA localization, and GTP binding to RhoA in MDA-MB-231 cells. These studies suggest that geranylgeranyl diphosphate synthase inhibitors are a novel approach to interfere with cancer cell migration.

Topics: Biosynthetic Pathways; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Diphosphonates; Female; Guanosine Triphosphate; Humans; Polyisoprenyl Phosphates; Protein Binding; Protein Transport; rhoA GTP-Binding Protein; Terpenes

Epidermal growth factor (EGF) exerts pleiotropic effects during oncogenesis, including the stimulation of cell migration and invasiveness. Although a number of traditional signaling proteins (e.g. Ras and Rho GTPases) have been implicated in EGF-stimulated cancer cell migration, less is known about the identity of those proteins functioning further downstream in this growth factor pathway. Here we have used HeLa carcinoma cells as a model system for investigating the role of tissue transglutaminase (TGase), a protein that has been linked to oncogenesis, in EGF-stimulated cancer cell migration and invasion. Treatment of HeLa cells with EGF resulted in TGase activation and its accumulation at their leading edges, whereas knocking down TGase expression, or treating cells with a TGase inhibitor, blocked EGF-stimulated cell migration and invasion. We show that EGF signaling through Ras and c-Jun N-terminal kinase is responsible for targeting TGase to the leading edges of cells and activating it. The requirement for EGF to properly localize and activate TGase can be circumvented by the expression of oncogenic Ras (G12V), whose ability to stimulate migration is also dependent on TGase. We further show that, in the highly aggressive breast cancer cell line MDAMB231, where EGF stimulation is unnecessary for migration and invasive activity, TGase is already at the leading edge and activated. These findings demonstrate that TGase plays a key role in cancer cell motility and invasiveness and represents a previously unappreciated participant in the EGF pathway that stimulates these processes in cancer cells.

Topics: Breast Neoplasms; Cell Membrane; Cell Movement; Cell Survival; Enzyme Activation; Epidermal Growth Factor; Female; Genes, ras; GTP-Binding Proteins; Guanosine Triphosphate; HeLa Cells; Humans; JNK Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Protein Glutamine gamma Glutamyltransferase 2; RNA, Small Interfering; Signal Transduction; Transglutaminases

Recently, we reported the identification of a novel gene named RBEL1 (Rab-like protein 1) and characterized its two encoded isoforms, RBEL1A and RBEL1B, that function as novel GTPases of Ras superfamily. Here we report the identification of two additional splice variants of RBEL1 that we have named RBEL1C and -D. All four RBEL1 isoforms (A, B, C, and D) have identical N termini harboring the Rab-like GTPase domains but contain variable C termini. Although all isoforms can be detected in both cytoplasm and nucleus, RBEL1A is predominantly cytoplasmic, whereas RBEL1B is mostly nuclear. RBEL1C and -D, by contrast, are evenly distributed between the cytoplasm and nucleus. Furthermore, all four RBEL1 proteins are also capable of associating with cellular membrane. The RBEL1 proteins also exhibit a unique nucleotide-binding potential and, whereas the larger A and B isoforms are mainly GTP-bound, the smaller C and D variants bind to both GTP and GDP. Furthermore, a regulatory region at amino acid position 236-302 immediately adjacent to the GTP-binding domain is important for GTP-binding potential of RBEL1A, because deletion of this region converts RBEL1A from predominantly GTP-bound to GDP-bound. RBEL1 knockdown via RNA interference results in marked cell growth suppression, which is associated with morphological and biochemical features of apoptosis as well as inhibition of extracellular signal-regulated kinase phosphorylation. Taken together, our results indicate that RBEL1 proteins are linked to cell growth and survival and possess unique biochemical, cellular, and functional characteristics and, therefore, appear to form a novel subfamily of GTPases within the Ras superfamily.

Topics: Alternative Splicing; Amino Acid Sequence; Apoptosis; Base Sequence; Breast Neoplasms; Cell Division; Cell Nucleus; Cytoplasm; Female; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; HeLa Cells; Humans; Isomerism; MAP Kinase Signaling System; Molecular Sequence Data; Protein Structure, Tertiary; ras Proteins; RNA, Small Interfering

The spatial organization of K-Ras proteins into nanoclusters on the plasma membrane is essential for high-fidelity signal transduction. The mechanism underlying K-Ras nanoclustering is unknown. We show here that K-Ras.GTP recruits Galectin-3 (Gal-3) from the cytosol to the plasma membrane where it becomes an integral nanocluster component. Importantly, we show that the cytosolic level of Gal-3 determines the magnitude of K-Ras.GTP nanoclustering and signal output. The beta-sheet layers of the Gal-3 carbohydrate recognition domain contain a hydrophobic pocket that may accommodate the farnesyl group of K-Ras. V125A substitution within this hydrophobic pocket yields a dominant negative Gal-3(V125A) mutant that inhibits K-Ras activity. Gal-3(V125A) interaction with K-Ras.GTP reduces K-Ras.GTP nanocluster formation, which abrogates signal output from the Raf/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK; MEK) pathway. Gal-3(V125A) negatively regulates cell growth and reduces cellular transformation. Thus, regulation of K-Ras nanocluster formation and signal output by Gal-3 critically depends on the integrity of the Gal-3 hydrophobic pocket. These results show that Gal-3 overexpression in breast cancer cells, which increases K-Ras signal output, represents oncogenic subversion of plasma membrane nanostructure.

Topics: Animals; Breast Neoplasms; Cell Adhesion; Cell Proliferation; Cells, Cultured; Cricetinae; Cytoplasm; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Galectin 3; Genes, ras; Guanosine Triphosphate; Humans; Immunoblotting; Immunoprecipitation; Kidney; Microscopy, Confocal; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-raf; Signal Transduction

Tea [Camellia sinensis (Theaceae)] intake is second only to water in terms of worldwide popularity as a beverage. The Green tea polyphenols have been shown to have a protective effect in prostate cancer in various pre-clinical animal models and has been reported to be effective in several other cancer types as well. An inverse association between the risk of breast cancer and the intake of green tea has also been reported in Asian Americans. Several epidemiological studies have shown that breast cancer progression is delayed in the Asian population that consumes green tea on regular basis. In this study, we report the effectiveness of green tea polyphenols (GTP) and its constituent Epigallocatechin Gallate (EGCG) in tumor regression using both in-vitro cell culture models and in vivo athymic nude mice models of breast cancer. The anti-proliferative effect of GTP and EGCG on the growth of human breast cancer MDA-MB-231 cell was studied using a tetrazolium dye-based (MTT) assay. Both GTP and EGCG treatment had the ability to arrest the cell cycle at G1 phase as assessed by flow cytometry. The expression of Cyclin D, Cyclin E, CDK 4, CDK 1 and PCNA were down regulated over the time in GTP and EGCG treated experimental group, compared to the untreated control group as evaluated by western blot analysis for cell cycle proteins, which corroborated the G1 block. Nude mice inoculated with human breast cancer MDA-MB-231 cells and treated with GTP and EGCG were effective in delaying the tumor incidence as well as reducing the tumor burden when compared to the water fed and similarly handled control. GTP and EGCG treatment were also found to induce apoptosis and inhibit the proliferation when the tumor tissue sections were examined by immunohistochemistry. Our results suggest that GTP and EGCG treatment inhibits proliferation and induce apoptosis of MDA-MB-231 cells in-vitro and in-vivo. All together, these data sustain our contention that GTP and EGCG have anti-tumor properties.

Topics: Administration, Oral; Animals; Anticarcinogenic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Catechin; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Female; Flavonoids; G1 Phase; Guanosine Triphosphate; Humans; Immunohistochemistry; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Phenols; Polyphenols; Proliferating Cell Nuclear Antigen; Tea; Xenograft Model Antitumor Assays

Rab family proteins are generally known as regulators of protein transport and trafficking. A number of Rab proteins have been implicated in cancer development and/or progression. Here we report the identification of a novel Rab-like protein, which we have named RBEL1 (Rab-like protein 1) for its higher similarity to the Rab subfamily members. We have characterized two isoforms of RBEL1 including the predominant RBEL1A and the less abundant RBEL1B that results from alternative splicing. Both isoforms harbor conserved N-terminal guanine trinucleotide phosphate (GTP) binding domains and, accordingly, are capable of binding to GTP. Both isoforms contain variable C termini and exhibit differential subcellular localization patterns. Unlike known Rabs that are mostly cytosolic, RBEL1B predominantly resides in the nucleus, whereas RBEL1A is localized primarily to the cytosol. Interestingly, a point mutation affecting RBEL1B GTP binding also alters the ability of mutant protein to accumulate in the nucleus, suggesting GTP binding potential to be important for RBEL1B nuclear localization. Our results also indicate that RBEL1A is overexpressed in about 67% of primary breast tumors. Thus, RBEL1A and RBEL1B are novel Rab-like proteins that localize in the nucleus and cytosol and may play an important role in breast tumorigenesis.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cytosol; Gene Expression Regulation, Neoplastic; Guanosine Triphosphate; Humans; Molecular Sequence Data; Nucleic Acid Hybridization; Point Mutation; Protein Binding; Protein Isoforms; Protein Structure, Tertiary; rab GTP-Binding Proteins; ras Proteins

Inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in the de novo synthesis of guanine nucleotides, is a major therapeutic target. A prototypic uncompetitive inhibitor of IMPDH, mycophenolic acid (MPA), is the active form of mycophenolate mofeteil (CellCept), a widely used immunosuppressive drug. We have found that MPA interacts with intracellular IMPDH in vivo to alter its mobility on SDS-polyacrylamide gels. MPA also induces a striking conformational change in IMPDH protein in intact cells, resulting in the formation of annular aggregates of protein with concomitant inhibition of IMPDH activity. These aggregates are not associated with any known intracellular organelles and are reversible by incubating cells with guanosine, which repletes intracellular GTP, or with GTPgammaS. GTP also restores IMPDH activity. Treatment of highly purified IMPDH with MPA also results in the formation of large aggregates of protein, a process that is both prevented and reversed by the addition of GTP. Finally, GTP binds to IMPDH at physiologic concentrations, induces the formation of linear arrays of tetrameric protein, and prevents the aggregation of protein induced by MPA. We conclude that intracellular GTP acts as an antagonist to MPA by directly binding to IMPDH and reversing the conformational changes in the protein.

Topics: Breast Neoplasms; Cell Line, Tumor; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; IMP Dehydrogenase; Mycophenolic Acid; Protein Conformation

AND-34, a 95-kDa protein with modest homology to Ras GDP exchange factors, associates with the focal adhesion protein p130Cas. Overexpression of AND-34 confers anti-estrogen resistance in breast cancer cell lines, a property linked to its ability to activate Rac. Here, we show that both the GDP exchange factor-like domain and the SH2 domain of AND-34 are required for Rac activation and for resistance to the estrogen receptor (ER) antagonist ICI 182,780. As phosphatidylinositol 3-kinase (PI3K) signaling can regulate Rac activation, we examined the effects of AND-34 on PI3K. Overexpression of AND-34 in MCF-7 cells increased PI3K activity and augmented Akt Ser(473) phosphorylation and kinase activity. Inhibition of PI3K with LY294002 or a dominant-negative p85 construct blocked AND-34-mediated Rac and Akt activation. Although R-Ras can activate PI3K, transfection with constitutively active R-Ras failed to induce Rac activation and AND-34 overexpression failed to induce R-Ras activation. Treatment of either vector-only or AND-34-transfected ZR-75-1 cells with ICI 182,780 markedly diminished ERalpha levels, suggesting that AND-34-induced anti-estrogen resistance is likely to occur by an ERalpha-independent mechanism. Treatment of a ZR-75-1 breast cancer cell line stably transfected with AND-34 plus 2 micromol/L LY294002 or 10 micromol/L NSC23766, a Rac-specific inhibitor, abrogated AND-34-induced resistance to ICI 182,780. Our studies suggest that AND-34-mediated PI3K activation induces Rac activation and anti-estrogen resistance in human breast cancer cell lines.

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromones; Crk-Associated Substrate Protein; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Estrogens; Focal Adhesions; Fulvestrant; Genes, Dominant; Genetic Vectors; GTP Phosphohydrolases; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Morpholines; Phosphatidylinositol 3-Kinases; Phosphorylation; Plasmids; Protein Binding; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; rac GTP-Binding Proteins; Receptors, Estrogen; Retinoblastoma-Like Protein p130; Serine; Signal Transduction; src Homology Domains; Transfection

While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor- and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.

Topics: Binding Sites; Breast Neoplasms; Carcinoma; cdc42 GTP-Binding Protein; Cell Cycle Proteins; Cell Line, Tumor; Cell Membrane; Clathrin-Coated Vesicles; Endosomes; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Humans; Nerve Tissue Proteins; p21-Activated Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Rho Guanine Nucleotide Exchange Factors; Wiskott-Aldrich Syndrome Protein, Neuronal

beta2-Chimerin is a member of the "non-protein kinase C" intracellular receptors for the second messenger diacylglycerol and the phorbol esters that is yet poorly characterized, particularly in the context of signaling pathways involved in proliferation and cancer progression. beta2-Chimerin possesses a C-terminal Rac-GAP (GTPase-activating protein) domain that accelerates the hydrolysis of GTP from the Rac GTPase, leading to its inactivation. We found that beta2-chimerin messenger levels are significantly down-regulated in human breast cancer cell lines as well as in breast tumors. Adenoviral delivery of beta2-chimerin into MCF-7 breast cancer cells leads to inhibition of proliferation and G(1) cell cycle arrest. Mechanistic studies show that the effect involves the reduction in Rac-GTP levels, cyclin D1 expression, and retinoblastoma dephosphorylation. Studies using the mutated forms of beta2-chimerin revealed that these effects were entirely dependent on its C-terminal GAP domain and Rac-GAP activity. Moreover, MCF-7 cells stably expressing active Rac (V12Rac1) but not RhoA (V14RhoA) were insensitive to beta2-chimerin-induced inhibition of proliferation and cell cycle progression. The modulation of G(1)/S progression by beta2-chimerin not only implies an essential role for Rac in breast cancer cell proliferation but also raises the intriguing possibility that diacylglycerol-regulated non-protein kinase C pathways can negatively impact proliferation mechanisms controlled by Rho GTPases.

Topics: Adenoviridae; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Diglycerides; G1 Phase; Guanosine Triphosphate; Humans; Mutation; Neoplasm Proteins; Phosphorylation; Protein Binding; Protein Kinase C; Protein Structure, Tertiary; rac GTP-Binding Proteins; Retinoblastoma Protein; RNA, Messenger; Signal Transduction; Time Factors

Our group recently identified Ras homolog member I (ARHI), a novel maternally imprinted tumor suppressor gene that encodes a 26 kDa GTP-binding protein with high homology to Ras and Rap. Unlike other Ras family members, ARHI exhibits several unusual structural and functional properties. ARHI contains a unique 34 amino-acid extension at the N-terminus, and differs from Ras in residues critical for GTPase activity and in its putative effector domain. Like Ras, ARHI can bind to GTP with high affinity but has low intrinsic GTPase activity. In addition, while Ras is an oncogene, ARHI functions as an inhibitor for cell growth. (32)Phosphorus labeling showed that ARHI is maintained in a constitutively activated GTP-bound state in resting cells, possibly because of impaired GTPase activity. ARHI is associated at the cell membrane through its prenylation at the C-terminal cysteine residue. Mutation of the conserved CAAX box at the C-terminus led to a loss of its membrane association and a decreased ability to inhibit cell growth. Conversion of Ser(51) to Asn decreased GTP binding and reduced ARHI's biological activity. Mutation of Ala(46) to Val increased the ability of ARHI to inhibit cell growth, associated with a further decrease of its intrinsic GTPase activity. Moreover, conversion of residues in ARHI that are conserved in the Ras family for GTPase activity partially restored the GTPase activity in ARHI. Most strikingly, deletion of ARHI's unique N-terminal extension nearly abolished its inhibitory effect on cell growth, suggesting its importance in ARHI's inhibitory function. Thus, ARHI is a unique Ras family member that retains basic small GTPase function, but exhibits many unusual features. In contrast to most other Ras family members, ARHI has a long N-terminal extension, modest GTPase activity, and constitutive GTP binding in resting cells. Furthermore, unlike the Ras oncogene, ARHI inhibits cell growth, and loss of its expression in cells may contribute to the development of breast and ovarian cancers.

Topics: Amino Acid Sequence; Breast Neoplasms; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; GTP Phosphohydrolases; Guanosine Triphosphate; Humans; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Ovarian Neoplasms; rho GTP-Binding Proteins; Sequence Alignment

The synthesis of a simplified analogue of the potent, cytotoxic tubulin-depolymerizing agent spongistatin 1, based on the AB spiroketal framework, is presented. The new structural analogue is an extension of a recently described spongistatin congener reported to disrupt microtubules in breast cancer cells in vitro and to alter the microtubule assembly reaction. Cytotoxicity data on the new structural analogue, as well as the parent congener, are reported. We found no significant cytotoxic or antitubulin activity with either compound.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cattle; Ethers, Cyclic; Guanosine Triphosphate; Humans; Lactones; Macrolides; Spiro Compounds; Structure-Activity Relationship; Tubulin; Tumor Cells, Cultured

In the present study, we addressed the question of a putative relevance of Rho proteins in tumour progression by analysing their expression on protein and mRNA level in breast tumours. We show that the level of RhoA, RhoB, Rac1 and Cdc42 protein is largely enhanced in all tumour samples analysed (n=15) as compared to normal tissues originating from the same individual. The same is true for (32)P-ADP-ribosylation of Rho proteins which is catalysed by Clostridium botulinum exoenzyme C3. Also the amount of Rho-GDI and ERK2 as well as the level of overall (32)P-GTP binding activity was tumour-specific elevated, yet to a lower extent than Rho proteins. Although the amount of Rho proteins was enhanced in tumours, most of them did not show changes in rho mRNA expression as compared to the corresponding normal tissue. Thus, elevated gene expression seems not to be the underlying mechanism of tumour-specific overexpression of Rho proteins. Sequence analysis of RhoA, RhoB, RhoC and Rac1 failed to detect any mutations in both the GTP-binding site and effector binding region. By analysing >50 tumour samples, the amount of RhoA-like proteins (i.e. RhoA, B, C), but not of Rac1, was found to significantly increase with histological grade and proliferation index. Rho protein expression was neither related to p53 nor to HER-2/neu oncogene status. Expression of rho mRNAs did not show a significant increase with histological grade. Overall the data show that (1) Rho proteins are overexpressed in breast tumours (2) overexpression is not regulated on the mRNA level (3) the expression level of RhoA-like proteins correlates with malignancy and (4) Rho proteins are not altered by mutation in breast tumours.

Topics: ADP Ribose Transferases; Blotting, Western; Breast; Breast Neoplasms; cdc42 GTP-Binding Protein; Disease Progression; DNA Mutational Analysis; Female; Gene Expression; Guanosine Triphosphate; Humans; Mitogen-Activated Protein Kinase 1; Mutation; Polymerase Chain Reaction; rac1 GTP-Binding Protein; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; RNA, Messenger

The Ras-GAP related protein IQGAP1 binds several proteins, including actin, calmodulin, E-cadherin and the Rho family GTPase Cdc42. To gain insight into its in vivo function, IQGAP1 was overexpressed in mammalian cells. Transfection of IQGAP1 significantly increased the levels of active, GTP-bound Cdc42, resulting in the formation of peripheral actin microspikes. By contrast, transfection of an IQGAP1 mutant lacking part of the GAP-related domain (IQGAP1deltaGRD) substantially decreased the amount of GTP-bound Cdc42 in cell lysates. Consistent with these findings, IQGAP1DeltaGRD blocked Cdc42 function in cells that stably overexpress constitutively active Cdc42 and abrogated the effect of bradykinin on Cdc42. In cells transfected with IQGAP1deltaGRD, bradykinin was unable to activate Cdc42, translocate Cdc42 to the membrane fraction, or induce filopodia production. IQGAP1deltaGRD transfection altered cellular morphology, producing small, round cells that closely resemble Cdc42-/- cells. Some insight into the mechanism was provided by in vitro analysis, which revealed that IQGAP1deltaGRD increased the intrinsic GTPase activity of Cdc42, thereby increasing the amount of inactive, GDP-bound Cdc42. These data imply that IQGAP1 has a crucial role in transducing Cdc42 signaling to the cytoskeleton.

Topics: Amino Acid Sequence; Animals; Breast Neoplasms; Carrier Proteins; Cattle; cdc42 GTP-Binding Protein; Cell Line; Cytoskeleton; Female; Guanosine Triphosphate; Humans; Molecular Sequence Data; ras GTPase-Activating Proteins; Recombinant Proteins; Saccharomyces cerevisiae; Schizosaccharomyces; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction; Transfection

Neurofibromin is a tumor suppressor protein, which is similar in function to the GTPase activating protein (GAP), p120GAP, in that it accelerates inactivation of Ras. Mutations in the NF1 gene cause neurofibromatosis type 1, NF1, an autosomal dominant disease with a diverse spectrum of clinical manifestations, including neurofibromas. Ras activation (GTP binding) is induced by the GTP exchange factor Sos and its inactivation is regulated through the GAPs (p120GAP and neurofibromin). Strikingly, neurofibromin was nearly absent in MB-231 human breast cancer cells and present in the remaining four cell lines studied, with higher levels in BT-474 and MB-453 than in MCF-7 and BT-20 cells, as tested with polyclonal antibodies to both the N-terminal as well as the C-terminal peptides. Coordinated with the near absence of neurofibromin, these cells also presented with much greater levels of P-MAPK and activated Ras. Further, RT-PCR analysis demonstrated the absence of expression of NF1 mRNA type I isoform only in the MB-231 cell lines. This result documents for the first time an altered NF1 expression at the protein and mRNA levels in MDA-MB-231 breast cancer cells.

Topics: Animals; Breast Neoplasms; Female; Guanosine Triphosphate; Humans; Mice; Mitogen-Activated Protein Kinases; Nerve Tissue Proteins; Neurofibromin 1; Phosphorylation; Rabbits; ras Proteins; RNA, Messenger; Tumor Cells, Cultured

Rac1 is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) that act as molecular switches to control cytoskeletal rearrangements and cell growth. Analogous to Ras, constitutively activating point mutations of Rac1 cause tumorigenic transformation of cell lines. However, there is no information about whether Rac1 is also mutated in vivo. After RT - PCR of Rac1, several clones of seven benign and 10 malignant breast cancer tissues as well as eight breast cancer cell lines were sequenced. Only single-nucleotide polymorphisms of Rac1 could be detected, and none of these corresponded to constitutively activating point mutations that have been used in cell lines for transformation. While sequencing Rac1 in breast tissues, a new Rac1 isoform with an insertion of 19 codons within the reading frame of Rac1 close to switch region II was identified and named Rac1b. The Rac1b protein acts like a fast cycling GTPase in GTP binding and hydrolysis assays. In Northern and Western blot experiments both Rac1 RNA and Rac1 protein had a significantly higher expression in breast cancer tissues compared to normal breast tissue samples. Immunohistochemical staining of Rac1 showed weak Rac1 expression in benign breast disease but high expression level in ductal carcinoma-in-situ, primary breast cancer, and lymph node metastases. In addition, breast tumor cells from patients with recurrent disease had Rac1 expression at the plasma membrane, suggesting activation of Rac1, in patients with aggressive breast cancer. Oncogene (2000).

Topics: Base Sequence; Breast Diseases; Breast Neoplasms; Cell Division; DNA Primers; Female; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Molecular Sequence Data; Polymorphism, Single Nucleotide; Protein Isoforms; rac1 GTP-Binding Protein; RNA, Messenger

We have shown previously that insulin promotes phosphorylation and activation of farnesyltransferase and geranylgeranyltransferase (GGTase) II. We have now examined the effect of insulin on geranylgeranyltransferase I in MCF-7 breast cancer cells. Insulin increased GGTase I activity 3-fold and augmented the amounts of geranylgeranylated Rho-A by 18%. Both effects of the insulin were blocked by an inhibitor of GGTase I, GGTI-286. The insulin-induced increases in the amounts of geranylgeranylated Rho-A resulted in potentiation of the Rho-A-mediated effects of lysophosphatidic acid (LPA) on a serum response element-luciferase construct. Preincubation of cells with insulin augmented the LPA-stimulated serum response element-luciferase activation to 12-fold, compared with just 6-fold for LPA alone (p < 0.05). The potentiating effect of insulin was dose-dependent, inhibited by GGTI-286 and not mimicked by insulin-like growth factor-1. We conclude that insulin activates GGTase I, increases the amounts of geranylgeranylated Rho-A protein, and potentiates the Rho-A-dependent nuclear effects of LPA in MCF-7 breast cancer cells.

Topics: Alkyl and Aryl Transferases; Breast Neoplasms; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Genes, Reporter; Guanosine Triphosphate; Humans; Hyperinsulinism; Insulin; Insulin-Like Growth Factor I; Luciferases; Lysophospholipids; Nuclear Proteins; Phosphorylation; Protein Processing, Post-Translational; Response Elements; rhoA GTP-Binding Protein; Serum Response Factor; Transcriptional Activation; Tumor Cells, Cultured

The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal-transducing Src/p21(ras)/Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c-Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti-progestins but also by anti-estrogens. In Cos-7 cells transfected with the B isoform of progesterone receptor (PRB), progestin activation of the MAP kinase pathway depends on co-transfection of ER. A transcriptionally inactive PRB mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PRB does not interact with c-Src but associates via the N-terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21(ras)/Erk pathway signals received from the agonist-activated PRB. These findings reveal a hitherto unrecognized cross-talk between ovarian hormones which could be crucial for their growth-promoting effects on cancer cells.

Topics: Animals; Breast Neoplasms; Carcinoma; COS Cells; CSK Tyrosine-Protein Kinase; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Estradiol; Gene Expression Regulation; Guanosine Triphosphate; Hormone Antagonists; Humans; Mitogen-Activated Protein Kinase 1; Point Mutation; Promegestone; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; src-Family Kinases; Tamoxifen; Transcriptional Activation; Tumor Cells, Cultured

Topics: 1-Phosphatidylinositol 4-Kinase; Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Division; Enzyme Inhibitors; Guanosine Triphosphate; Humans; Inositol 1,4,5-Trisphosphate; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Tumor Cells, Cultured; Type C Phospholipases

Computer-assisted structure analysis indicated (+)-discodermolide, a polyhydroxylated alkatetraene lactone marine natural product, was an antimitotic compound, and we confirmed this prediction. Previous work had shown an accumulation of discodermolide-treated cells in the G2/M portion of the cell cycle, and we have now found that discodermolide arrests Burkitt lymphoma cells in mitosis. Discodermolide-treated breast carcinoma cells displayed spectacular rearrangement of the microtubule cytoskeleton, including extensive microtubule bundling. Microtubule rearrangement that occurred with 10 nM discodermolide required 1 microM taxol. Discodermolide had equally impressive effects on tubulin assembly in vitro. Near-total polymerization occurred at 0 degree C with tubulin plus microtubule-associated proteins (MAPs) under conditions in which taxol at an identical concentration was inactive. Without MAPs and/or without GTP, tubulin assembly was also more vigorous with discodermolide than with taxol under every reaction condition examined. Discodermolide-induced polymer differed from taxol-induced polymer in that it was completely stable at 0 degree C in the presence of high concentrations of Ca2+. In a quantitative assay designed to select for agents more effective than taxol in inducing assembly, discodermolide had an EC50 value of 3.2 microM versus 23 microM for taxol.

Topics: Alkanes; Animals; Antineoplastic Agents; Breast Neoplasms; Burkitt Lymphoma; Calcium; Carbamates; Cell Division; Cell Line; Dose-Response Relationship, Drug; Female; Fluorescent Antibody Technique, Indirect; Guanosine Triphosphate; Humans; Kinetics; Lactones; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Paclitaxel; Porifera; Pyrones; Time Factors; Tubulin; Tumor Cells, Cultured

The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor.

Topics: Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; CSK Tyrosine-Protein Kinase; Enzyme Activation; Estradiol; Female; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nuclear Proteins; Phosphoproteins; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Proto-Oncogene Proteins pp60(c-src); ras-GRF1; Receptors, Estradiol; Repressor Proteins; Signal Transduction; src Homology Domains; src-Family Kinases; Tumor Cells, Cultured

LU103793 (NSC D-669356) is a new synthetic derivative of Dolastatin 15, an antiproliferative compound which was isolated from the mollusk Dolabella auricularia. Like Dolastatin 15, LU103793 is highly cytotoxic in vitro (IC50 = 0.1 nM). To investigate the mechanism of action of LU103793, we used a combination of biochemical and cellular methods. Turbidity assays with bovine brain microtubules demonstrated that LU103793 inhibits microtubule polymerization in a concentration-dependent manner (IC50 = 7 microM). Treatment with this compound also induced depolymerization of preassembled microtubules. Cell cycle analysis of tumor cell lines treated with LU103793 indicated a block in the G2-M phase. At the cellular level, it induced depolymerization of microtubules in interphase cells and development of abnormal spindles and chromosome distribution in mitotic cells. Although these effects are very similar to the cellular alterations caused by vinblastine, LU103793 does not inhibit vinblastine binding to unpolymerized tubulin in vitro. Our results suggest that LU103793 exerts its cytotoxic activity primarily through disruption of microtubule organization.

Topics: Adenocarcinoma; Amino Acid Sequence; Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Division; Chromosomes, Human; Colonic Neoplasms; Drug Interactions; Guanosine Triphosphate; HeLa Cells; Humans; Hydrolysis; Microtubules; Mitosis; Molecular Sequence Data; Neoplasms; Oligopeptides; Tubulin; Tumor Cells, Cultured; Vinblastine

Several G-proteins (GTP-binding proteins) were identified by SDS/PAGE in the cytosol (105,000 g supernatant) and membrane fractions of the oestrogen-dependent human mammary-tumour cell line ZR-75-1. These proteins, with molecular masses in the range 18-29 kDa, specifically bind [alpha-32P]GTP, which can be displaced by unlabelled GTP, GDP and their non-hydrolysable analogues guanosine 5'-[delta-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]), but not by GMP, ATP, ADP, AMP and other unrelated nucleotides. The apparent dissociation constant for GTP was approx. 2 x 10(-8)M. Homogenization of ZR-75-1 cells in high-salt buffer (1 M-KCl), and successive washing of the membrane fraction, suggested that, among the major G-proteins found, the 18 kDa protein is predominantly soluble, whereas the 27-29 kDa complex is primarily bound to the membrane fraction under the experimental conditions employed. Possible translocation of these G-proteins between membrane and cytosol was analysed. No redistribution of the 27-29 kDa complex was observed, whereas GTP[S] in the presence of Mg2+ caused apparent translocation of the 18 kDa protein to the membrane fraction. This effect was specific for GTP and stable GTP analogues, whereas GDP, GMP, ATP, ADP, AMP and other unrelated nucleotides were ineffective. GTP[S] and guanosine 5'-[beta gamma-imido]-triphosphate (p[NH]ppG) were equally potent (apparent Kd approximately 5 x 10(-6)M), whereas GTP was rather weak. The nucleotide effect is temperature-, time- and concentration-dependent. The translocation process was reversible, slow, and reached its maximum between 30 and 60 min at 37 degrees C. The apparent translocation of this small G-protein from the cytosol to the membrane fraction, and the specific effect of GTP analogues, suggest that this process may have functional significance in mammary-tumour cells.

Topics: Biological Transport; Breast Neoplasms; Cell Membrane; Cytosol; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Kinetics; Magnesium; Molecular Weight; Temperature; Thionucleotides; Tumor Cells, Cultured

The effect of the nucleoside anti-metabolite tiazofurin (TR) was examined on the growth and phenotypic alterations of MCF-7 breast cancer and HBL-100 normal breast cell lines. TR was shown to inhibit MCF-7 cell growth. This inhibition could be reversed by exogenous addition of guanosine. The anti-proliferative effect of TR is accompanied by phenotypic alterations that include lipid accumulation and an increase in alkaline phosphatase activity. In contrast to MCF-7 cells, the HBL-100 breast milk derived cell line is relatively resistant to inhibition by TR. Alkaline phosphatase is not affected by TR and untreated cells accumulate lipid droplets, similar to TR-treated MCF-7 cells. Determination of GTP and ATP pools in both cell lines revealed that TR markedly reduces GTP content in MCF-7 cells. In HBL-100 cells, TR induces only a small decrease in GTP and does not affect ATP levels. The prototypic IMP dehydrogenase inhibitor, mycophenolic acid (MA), markedly inhibits HBL-100 cell growth, similarly to its effect on MCF-7 breast cancer cells. These findings may suggest differential metabolism of TR in MCF-7 and HBL-100 cells.

Topics: Adenosine Triphosphate; Antimetabolites, Antineoplastic; Breast Neoplasms; Cell Division; Cell Line; Female; Guanosine Triphosphate; Humans; Milk, Human; Mycophenolic Acid; Ribavirin; Ribonucleosides; Tumor Cells, Cultured

High-performance liquid chromatography-purified 125I-vasoactive intestinal peptide (VIP) bound to T-47D human breast cancer cells in a specific, saturable, and reversible manner. Scatchard plots were compatible with the presence of one class of VIP receptors with high affinity (Kd = 4.5 X 10(-10) M VIP, and Bmax = 293 fmol/mg protein). The neuropeptide and its natural analogues inhibited the binding of 125I-VIP and stimulated cyclic AMP (cAMP) generation in T-47D cells 96-fold (EC50 = 7 X 10(-10) M VIP), in the following order of potency: VIP greater than helodermin greater than human peptide with N-terminal histidine and C-terminal methionine greater than human pancreatic growth hormone-releasing factor greater than human secretin. In contrast, 125I-VIP binding was not displaced by pancreatic glucagon, human oxyntomodulin, truncated glucagon-like peptide-1, glucagon-like peptide-2, the somatostatin analogue SMS 201-995, gastric inhibitory peptide, and a series of steroid hormones or peptides unrelated to VIP. VIP also increased cAMP generation in seven other human breast cancer cell lines: H4-66B, HSL 53, HSL 78, MCF 7, MDA-MB231, T-47D2, and ZR75-1. Adenylate cyclase activity rose from 72.2 +/- 14 to 1069 +/- 66 pmol cAMP/min mg protein after the addition of 10(-7) M VIP to T-47D plasma membranes. In agreement with our pharmacological results and the Scatchard analysis of the binding data, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized receptor in the T-47D membranes permitted identification of one autoradiographic band with a molecular weight of 69,000. The sensitivity of the Mr 69,000 binding site to GTP and low doses of VIP implies that in T-47D cells, this component constitutes the membrane domain involved in the functional regulation of adenylate cyclase by VIP receptors. Our results indicate a role for the VIP receptor-cAMP system in human breast cancer cells.

Topics: Adenylyl Cyclases; Breast Neoplasms; Cell Line; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Female; Guanosine Triphosphate; Humans; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Succinimides; Vasoactive Intestinal Peptide

Topics: Adenosine Triphosphate; Breast Neoplasms; Cell Survival; Dose-Response Relationship, Drug; Female; Guanosine; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Ketone Oxidoreductases; Mitosis; Mycophenolic Acid; Phenotype; Tumor Cells, Cultured

Monoclonal antibody D5, raised against cytosolic human estrogen receptor (ER) reacts with p29, a receptor-associated cytoplasmic serine phosphoprotein which does not bind steroid, While p29 selectively binds GTP and to a lesser extent ATP, in vitro GTP binding does not result in p29 phosphorylation. Under ER activating conditions, p29 associates with cytosolic ER; GTP, ATP and sodium molybdate block formation of immunoprecipitable p29-ER complexes. Nucleotide binding data suggest a role for p29 in the estrogen response machinery, possibly at the level of phosphate or nucleotide metabolism.

Topics: Adenosine Triphosphate; Biomarkers, Tumor; Breast Neoplasms; Cell Line; Female; Guanosine Triphosphate; Heat-Shock Proteins; Humans; Myometrium; Phosphoproteins; Phosphorylation; Receptors, Estrogen

An unusual isozyme of lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27), LDHk, has been described in cells transformed by the Kirsten murine sarcoma virus (KiMSV). This isozyme appears to contain one or more subunits encoded by the transforming gene of KiMSV and is readily distinguished from other isozymes of LDH. Specifically, it is more basic than other LDH isozymes, has an apparent subunit structure of (35,000)4(22,000)1, is essentially inactive if assayed under a normal atmosphere, and is strongly inhibited by GTP and various related compounds. We have examined human cancer and normal tissue controls for expression of an activity like LDHk. In 11 out of 16 human carcinomas, LDHk activity was increased 10- to 500-fold over the level seen in adjoining nontumor tissue. In contrast, other LDH isozymes were increased by only 2- to 5-fold.

Topics: Aerobiosis; Breast Neoplasms; Colonic Neoplasms; Female; Guanosine Triphosphate; HeLa Cells; Humans; Isoenzymes; L-Lactate Dehydrogenase; Neoplasms; Reference Values