Inhibition Of Rho GTPases Research Articles

Neuronal Apoptosis Induced by Selective Inhibition of Rac GTPase versus Global Suppression of Rho Family GTPases Is Mediated by Alterations in Distinct Mitogen-activated Protein Kinase Signaling Cascades

Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad.

Molecular pathways: targeting the kinase effectors of RHO-family GTPases.

RHO GTPases, members of the RAS superfamily of small GTPases, are adhesion and growth factor-activated molecular switches that play important roles in tumor development and progression. When activated, RHO-family GTPases such as RAC1, CDC42, and RHOA, transmit signals by recruiting a variety of effector proteins, including the protein kinases PAK, ACK, MLK, MRCK, and ROCK. Genetically induced loss of RHO function impedes transformation by a number of oncogenic stimuli, leading to an interest in developing small-molecule inhibitors that either target RHO GTPases directly, or that target their downstream protein kinase effectors. Although inhibitors of RHO GTPases and their downstream signaling kinases have not yet been widely adopted for clinical use, their potential value as cancer therapeutics continues to facilitate pharmaceutical research and development and is a promising therapeutic strategy.

Abstract A56: Expression of CXCR4 is highly dynamic in Ewing sarcoma and its upregulation in response to microenvironmental stress promotes cell migration and invasion

Abstract Background and Objective: Ewing sarcoma (ES) is the second most common malignant bone tumor in pediatric patients. Although the primary cause of tumor-related death in ES is relapse at distant sites, the mechanisms underlying metastatic relapse remain to be fully elucidated. In the current study we are investigating the role of the CXCR4/CXCL12 chemokine axis as a mediator of ES metastasis. Methods: Expression of CXCR4 transcript and surface protein were quantified using qRT-PCR and flow cytometry. Label-retaining, putative cancer stem cells were isolated 10-12 days following PKH-dye incorporation into cell membranes. Chemotactic migration and invasion to SDF-1α were evaluated in unmodified cells and in cells following CXCR4 knockdown or exposure to the CXCR4 antagonist AMD3100. Small molecule inhibitors of Rho-GTPases were used to assess the contribution of these proteins to CXCR4-dependent migration. Results: Baseline expression of CXCR4 transcript and surface protein varied widely among ES cell lines. Immunohistochemical staining of primary tumors also revealed marked variability of CXCR4, ranging from completely undetectable to high-level expression. Interestingly, CXCR4 transcript was increased in slowly proliferating (PKH-dye-retaining) TC71 and A673 cells implicating CXCR4 as a potential marker of ES cancer stem cells. To determine if CXCR4 expression is static or responsive to changes in the microenvironment we measured transcript levels and the frequency of CXCR4+ cells under different experimental conditions. Significantly, serum deprivation, hypoxia and growth constraint (cell confluence) all resulted in upregulation of CXCR4 mRNA and CXCR4+ cell frequency in each of 4 different ES cell lines. This upregulation of CXCR4 was rapidly reversed upon removal of each of the stressors indicating the plastic and dynamic nature of CXCR4 regulation. In addition, within 3 weeks of FACS sorting, TC-32 ES cells that had been sorted into CXCR4+ and CXCR4- populations reverted to their baseline state, demonstrating that CXCR4+ cells can change into CXCR4- cells and vice versa. Functionally, CXCR4+ ES cells migrated and invaded towards SDF-1α, the ligand for CXCR4 and this chemotactic migration and invasion were impeded pharmacologically by AMD3100, and genetically by CXCR4 knockdown. Interestingly, we observed complete abrogation of invasion through Matrigel in CXCR4 knockdown and AMD3100 treated cells suggesting a critical role for CXCR4 in mediating ES cell invasion through basement membranes. To begin to define the mechanism of CXCR4-mediated invasion we exposed ES cells to small molecule inhibitors of the Rho-GTPases CDC42 and RAC1. Importantly, sub-cytotoxic doses of both compounds suppressed ES cell migration towards SDF-1α. Conclusions: We have demonstrated the highly dynamic nature of CXCR4 expression in ES cells and have shown that this molecular plasticity allows ES to switch back and forth between CXCR4- and CXCR4+ states in response to changes in the microenvironment. Significantly, ES cells that express high levels of CXCR4 are highly migratory and invasive in response to chemotactic signaling from SDF-1α and preliminary data suggest that Rho-GTPases CDC42 and/or RAC1 mediate this aggressive phenotype. We propose a model in which stressors in the microenvironment induce ES cells to upregulate CXCR4 thus contributing to cellular migration and invasion. Ongoing work is testing whether CXCR4+ ES cells are more metastatic than CXCR4- cells in vivo. The CXCR4/CXCL12 axis may thus be a novel therapeutic target for the prevention of ES metastasis. Citation Format: Melanie A. Krook, Lauren A. Nicholls, Christopher A. Scannell, Rashmi Chugh, Dafydd G. Thomas, Elizabeth R. Lawlor. Expression of CXCR4 is highly dynamic in Ewing sarcoma and its upregulation in response to microenvironmental stress promotes cell migration and invasion. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A56.

Targeting Fyn in Ras-transformed cells induces F-actin to promote adherens junction-mediated cell-cell adhesion.

Fyn, a member of the Src family kinases (SFK), is an oncogene in murine epidermis and is associated with cell-cell adhesion turnover and induction of cell migration. Additionally, Fyn upregulation has been reported in multiple tumor types, including cutaneous squamous cell carcinoma (cSCC). Introduction of active H-Ras(G12V) into the HaCaT human keratinocyte cell line resulted in upregulation of Fyn mRNA (200-fold) and protein, while expression of other SFKs remained unaltered. Transduction of active Ras or Fyn was sufficient to induce an epithelial-to-mesenchymal transition in HaCaT cells. Inhibition of Fyn activity, using siRNA or the clinical SFK inhibitor Dasatinib, increased cell-cell adhesion and rapidly (5-60 min) increased levels of cortical F-actin. Fyn inhibition with siRNA or Dasatinib also induced F-actin in MDA-MB-231 breast cancer cells, which have elevated Fyn. F-actin co-localized with adherens junction proteins, and Dasatinib-induced cell-cell adhesion could be blocked by Cytochalasin D, indicating that F-actin polymerization was a key initiator of cell-cell adhesion through the adherens junction. Conversely, inhibiting cell-cell adhesion with low Ca(2+) media did not block Dasatinib-induced F-actin polymerization. Inhibition of the Rho effector kinase ROCK blocked Dasatinib-induced F-actin and cell-cell adhesion, implicating relief of Rho GTPase inhibition as a mechanism of Dasatinib-induced cell-cell adhesion. Finally, topical Dasatinib treatment significantly reduced total tumor burden in the SKH1 mouse model of UV-induced skin carcinogenesis. Together these results identify the promotion of actin-based cell-cell adhesion as a newly described mechanism of action for Dasatinib and suggest that Fyn inhibition may be an effective therapeutic approach in treating cSCC.

Rho GTPases Control Ciliary Epithelium Cells Proliferation and Progenitor Profile Induction In Vivo

Rho GTPases play a central role in actin-based cytoskeleton reorganization and regulate multiple signaling pathways that control gene transcription, cell survival, and proliferation. We investigated the effect of Rho GTPases on cell cycle regulation and progenitor genes expression on mouse ciliary epithelium (CE), a potential source of progenitor/stem cells in the adult retina. Rho GTPases were activated by intraocular injection of lysophosphatidic acid and inactivated by Clostridium difficile Toxin A (general Rho GTPase inhibitor), NSC23766 (Rac1 activation inhibitor), or Y27632 (Rho-associated protein kinase [ROCK] inhibitor). Thereafter, we assayed for RhoA, RhoB, and Rac1 protein localization in CE cells. Proliferation was examined by the expression levels of cell cycle regulators p27(kip), p16(INK4a), and Ki67 and the effects on progenitors by determining the changes in Pax6 and Chx10 progenitor markers expression. All GTPases investigated were expressed in mouse CE cells. Activation increased the coexpression of Pax6 and Chx10, but had no significant effect on the proliferation of CE cells. In contrast, Rho GTPases inactivation increased cell proliferation and potentiated the proliferative effect of growth factors. Specific inactivation of Rac1 or ROCK increased the levels of Ki67 and decreased the expression of the cell cycle inhibitors p27(kip) and p16(INK4a). This study reports that Rho GTPase modulation (activation and inactivation) controls the expression of retinal progenitor genes and proliferation, respectively, in the adult ciliary epithelial progenitor/stem cells of rodent eyes. The modulation of these two different mechanisms (proliferation and reprogramming) may provide a potential new approach in retinal repair.

Trkb Signaling in Pericytes Is Required for Cardiac Microvessel Stabilization

Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb −/−) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb−/− embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.

Effects of intravitreal injection of a Rho-GTPase inhibitor (BA-210), or CNTF combined with an analogue of cAMP, on the dendritic morphology of regenerating retinal ganglion cells

In adult rats, intravitreal injections of the Rho-GTPase inhibitor C3 transferase (BA-210), or a cocktail of recombinant ciliary neurotrophic factor (CNTF) and a cyclic AMP analogue (CPTcAMP), increase retinal ganglion cell (RGC) survival and axonal regeneration. Here we examined whether these treatments also affect the dendritic architecture of regrowing RGCs. In Fischer F344 rats, one optic nerve was cut and an autologous peripheral nerve graft was sutured onto it. Rats then received intravitreal injections (4 μl) of saline, BA-210 or CNTF + CPTcAMP four and eleven days after the PN graft surgery. After 5 weeks, regenerating RGCs were retrogradely labelled with fluorogold (FG) and in living wholemounts the dendritic trees of FG labelled RGCs were visualised by intracellular injection of 2% Lucifer Yellow. Injection of BA-210 or CNTF + CPTcAMP resulted in significantly more regenerating RGCs with abnormal dendritic morphologies, including abnormally long looping processes. Compared to saline-injected regenerating controls, RGCs in BA-210 injected eyes had significantly smaller dendritic field areas and sparser dendrites, while in CNTF + CPTcAMP injected eyes there was increased branching of more distal dendrites. While both intraocular treatments enhance RGC axonal regrowth, they also induce significant changes in RGC dendritic morphology. It remains to be determined if such changes alter the function of the regenerating neuronal population.

Activation of Rac-1 and RhoA Contributes to Podocyte Injury in Chronic Kidney Disease

Rho-family GTPases like RhoA and Rac-1 are potent regulators of cellular signaling that control gene expression, migration and inflammation. Activation of Rho-GTPases has been linked to podocyte dysfunction, a feature of chronic kidney diseases (CKD). We investigated the effect of Rac-1 and Rho kinase (ROCK) inhibition on progressive renal failure in mice and studied the underlying mechanisms in podocytes. SV129 mice were subjected to 5/6-nephrectomy which resulted in arterial hypertension and albuminuria. Subgroups of animals were treated with the Rac-1 inhibitor EHT1846, the ROCK inhibitor SAR407899 and the ACE inhibitor Ramipril. Only Ramipril reduced hypertension. In contrast, all inhibitors markedly attenuated albumin excretion as well as glomerular and tubulo-interstitial damage. The combination of SAR407899 and Ramipril was more effective in preventing albuminuria than Ramipril alone. To study the involved mechanisms, podocytes were cultured from SV129 mice and exposed to static stretch in the Flexcell device. This activated RhoA and Rac-1 and led via TGFβ to apoptosis and a switch of the cells into a more mesenchymal phenotype, as evident from loss of WT-1 and nephrin and induction of α-SMA and fibronectin expression. Rac-1 and ROCK inhibition as well as blockade of TGFβ dramatically attenuated all these responses. This suggests that Rac-1 and RhoA are mediators of podocyte dysfunction in CKD. Inhibition of Rho-GTPases may be a novel approach for the treatment of CKD.

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<i>Circulation</i> Editors’ Picks

A novel small-molecule screening strategy identifies mitoxantrone as a RhoGTPase inhibitor

RhoGTPases are GDP/GTP molecular switches that control a wide variety of cellular processes, thereby contributing to many diseases, including cancer. As a consequence, there is great interest in the identification of small-molecule inhibitors of RhoGTPases. In the present paper, using the property of GTP-loaded RhoGTPases to bind to their effectors, we describe a miniaturized and robust assay to monitor Rac1 GTPase activation that is suitable for large-scale high-throughput screening. A pilot compound library screen revealed that the topoisomerase II poison MTX (mitoxantrone) is an inhibitor of Rac1, and also inhibits RhoA and Cdc42 in vitro. We show that MTX prevents GTP binding to RhoA/Rac1/Cdc42 in vitro. Furthermore, MTX strongly inhibits RhoGTPase-mediated F-actin (filamentous actin) reorganization and cell migration. Hence, we report a novel biochemical assay yielding the identification of RhoGTPase inhibitors and we present a proof-of-concept validation with the identification of MTX as a novel pan-RhoGTPase inhibitor.

Atypical PKC, regulated by Rho GTPases and Mek/Erk, phosphorylates Ezrin during eight-cell embryocompaction

Phosphorylation of Ezrin T567 plays an important role in eight-cell embryo compaction. Yet, it is not clear how Ezrin phosphorylation is regulated during embryo compaction. Here, we demonstrated that inhibition of Mek/Erk or protein kinase C (PKC) signaling reduced the phosphorylation level of Ezrin T567 in eight-cell compacted embryos. Interestingly, the Rho GTPase inhibitor C3-transferase caused basolateral enrichment of atypical PKC (aPKC), as well as basolateral shift of phosphorylated Ezrin, suggesting aPKC may be a key regulator of Ezrin phosphorylation. Moreover, inhibition of PKC, but not Mek/Erk or Rho GTPases, affected the maintenance of Ezrin phosphorylation in compacted embryos. We further identified that aPKC is indeed required for Ezrin phosphorylation in eight-cell embryos. Taken together, Rho GTPases facilitate the apical distribution of aPKC and Ezrin. Subsequently, aPKC and Mek/Erk work together to promote Ezrin phosphorylation at the apical region, which in turn mediates the apical enrichment of filamentous actin, stabilizing the polarized apical region and allowing embryo compaction. Our data also suggested that aPKC might be the Ezrin kinase during eight-cell embryo compaction.

Transfection in the third dimension

An understanding of parameters that modulate gene transfer in 3-D will assist in the formation of gene delivery systems and scaffolds, which can mediate efficient non-viral delivery for guiding in vivo tissue regeneration and therapy. We have previously demonstrated the cell area and length, integrin expression, and RhoGTPase mediated signalling to be pivotal parameters that guide gene transfer to mouse mesenchymal stem cells (mMSCs) cultured in 2-D and are modulated by ECM proteins. In this study, we were interested in determining if cationic polymer mediated gene transfer to cells seeded in 3-D would occur through different mechanisms as compared to those seeded in 2-D. In particular, we examined the endocytosis pathways used to internalize polyplexes, and the role of cytoskeletal dynamics and RhoGTPases in non-viral gene transfer for cells seeded in 2-D and 3-D. Inhibition of clathrin- and caveolae-mediated endocytosis resulted in a more drastic decrease in overall transgene expression for cells seeded in 3-D than for those in 2-D. In addition, polyplex internalization was only significantly decreased in 3-D when clathrin-mediated endocytosis was inhibited, while caveolae-mediated endocytosis inhibition for cells seeded in 2-D resulted in the strongest polyplex internalization inhibition. Actin and microtubule polymerization affected 2-D and 3-D transfection differently. Microtubule depolymerization enhanced transgene expression in 2-D, but inhibited transgene expression in 3-D. Lastly, inhibition of RhoGTPases also affected 2-D and 3-D transfection differently. The inhibition of ROCK effectors resulted in a decrease of transgene expression and internalization for cells seeded in 3-D, but not in 2-D, and the inhibition of the effector PAK1 resulted in an increase of transgene expression for both 2-D and 3-D. Overall, our study suggests that the process of gene transfer occurs through different mechanisms for cells seeded in 2-D compared to those seeded in 3-D.

A FRET-Based Approach for Quantitative Evaluation of Forskolin-Induced Pendrin Trafficking at the Plasma Membrane in Bronchial NCI H292 Cells

Background: Human pendrin (SLC26A4, PDS) is an integral membrane protein acting as an electroneutral anion exchanger. Loss of function mutations in pendrin protein cause Pendred syndrome, a disorder characterized by sensorineural deafness and a partial iodide organification defect that may lead to thyroid goiter. Additionally, pendrin up-regulation could play a role in the pathogenesis of several diseases including bronchial asthma and chronic obstructive pulmonary disease (COPD). Therefore, monitoring the plasma membrane abundance and trafficking of pendrin in the context of a living cell is crucially important. Methods: Trafficking of pendrin to the plasma membrane was monitored by fluorescence resonance energy transfer (FRET), a physical phenomenon occurring between two fluorophores (the FRET donor and acceptor) located in close spatial proximity. Because the efficiency of the energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, FRET is extremely sensitive to small changes in distance between the donor and acceptor and is therefore a powerful tool to determine protein-protein interactions. Results: FRET studies revealed that forskolin-induced cAMP production is associated with a significant increase of pendrin expression at plasma membrane, which is paralleled by a decrease in intracellular pH. Pendrin transposition to the membrane is accompanied with a partial depolymerization of actin cytoskeleton via Rho-GTPase inhibition. Conclusion: Trafficking to the plasma membrane is critical in the regulation of pendrin activity. Therefore, reliable tools for monitoring and quantifying this phenomenon are highly desirable.

New clues for organogenesis

Organogenesis is a conventional research area in developmental biology. Recent research progresses on stem cell, epigenetics, no-coding RNA and other newpropertiesofmulticellularorganismshavetransformed the organogenesis research as a frontier of life science. The collection of six research papers in this issue aims to cover timely developments in the research area of organogenesis from basic mechanism analysis to applied medical potential. In the first article, Dr Wu’s group reported that knockdown of the members of the cadherin cell adhesion superfamily proteins, protocadherin (Pcdh) clusters, resulted in dendritic simplification and spine defects in vivo and in cultured primary hippocampal neurons in vitro. Furthermore, the knockdown of the Pcdha cluster resulted in the activation of two cell-adhesion kinases (Pyk2 and Fak) and inhibition of Rho GTPases that is essential for neuronal morphogenesis and synaptic plasticity. The authors propose that Pcdh clusters and CAKs play important roles in dendritic development and spine elaboration. In the second article, Dr Rottier’s group analyzed the function of a transcription factor (TF) Sox2 in regulating the initial patterning of mouse primitive gut. The authors showed that ectopic expression of Sox2 in the posterior region of the primitive gut caused anteriorization of the gut toward a gastriclike phenotype. The authors further showed that Sox2 activated the foregut transcriptional program even in the condition of sustained co-expression of an endogenous TF Cdx2, which is recognized as a suppressor of anteriorization. They conclude that the balance between Sox2 and Cdx2 function is essential for proper specification of the primitive gut, and that Sox2 could reprogram the initial patterning of the primitive gut. It is known that a muscle-specific TF myogenin (Myog) and an MAP kinase (p38a) are required during the process of myogenesis. By comparing the expression profiling of Myog-activated alone with that of p38a activated alone in myogenesis with microarray approach, Dr Dilworth’s group showed that Myog and p38a played different roles in muscle differentiation. The authors found an un

Abstract 07: The effect of genetic variants on the relationship between statins and breast cancer in the Women's Health Initiative Observational Study.

Abstract Background: Statins are the most widely prescribed and most effective cholesterol-lowering drugs used in the United States. Statins are a logical candidate for cancer chemoprevention in that they have multiple cellular effects other than lowering cholesterol, including: inhibition of rho GTPases, induction of apoptosis, and decreasing markers of chronic inflammation. A chemopreventive effect of statins on breast cancer has been suggested, however there is significant variation in inter-individual response to statins, and underlying genetic differences may modify the effect of statins on cancer risk. The purpose of this analysis was to evaluate whether there is an interaction between genes modifying statin metabolism and statin use in relationship with breast cancer risk in a case-control study nested within the Women's Health Initiative (WHI) Observational Study (OS). Methods: Genome Wide Association Study (GWAS) data on 30,380 SNPS were available for 1,683 breast cancer cases and 1,683 one-to-one matched controls from the WHI and were included in Cancer Genetic Markers of Susceptibility (CGEMS). Matching criteria were: age at screening, enrollment date, race/ethnicity (all were White), hysterectomy status at baseline and history of breast cancer at baseline. Statin use, sociodemographic information and potentially confounding variables were evaluated at baseline and breast cancer diagnoses were centrally adjudicated. We examined interactions between baseline statin use (yes or no) and 22 SNPs in or near 9 candidate lipid metabolism genes using a conditional logistic model adjusted for features at baseline associated with case/control status. We also examined interactions between statin use and all available SNPs from the GWAS after adjustment for significant features. Results: There were two SNPs out of the 22 SNPs in or near the lipid genes of interest that were nominally significant; rs1529711 in the CARM1 gene [near candidate gene SMARC4, minor allele frequency (MAF) 15%], Pint=0.04, and rs9282564 in the ABCB1 gene (MAF 10%), Pint=0.01. When all GWAS SNPs were examined for interactions with statin use, 34 SNPs achieved statistical significance using a 5% false discovery rate. The GWAS SNP with the greatest evidence for interaction with statin use on breast cancer risk was rs2875218 (Pint=0.0000076). Conclusion: Two SNPs (rs1529711 and rs9282564) near candidate genes (SMARC4 and ABCB1, respectively) showed trends as effect modifiers of statins on breast cancer risk. SMARC4 is a SWI/SNF related, matrix associated, actin dependent regulator of chromatin, and was identified as a candidate effect modifier in a genome-wide study of statin induced myopathy (Link et al., NEJM, 2008). ABCB1 encodes a large transmembrane protein integral to the blood-brain barrier and functions as a drug-transport pump between brain and blood which attenuated LDL-C reduction in a study by Newman and Hulley (JAMA 1996). From the genome-wide analyses, a SNP (rs2875218) located between PRDX3P3 and FREM2 on chromosome 13 was strongly implicated as interacting with statin use on breast cancer risk. Future analyses will examine tagSNPs covering genomic regions of interest including candidate genes and loci identified through this genome-wide examination of SNP x Statin interactions. We will also explore the associations by statin subtype (lipophilic, hydrophilic). Genetic associations such as these, if confirmed, may have implications in terms of personalized medicine, given that statin efficacy in relation to breast cancer risk may be increased or decreased in individuals dependent on inherited genetic polymorphisms. Citation Format: Allison M. Jay, Cathryn H. Bock, Gregory Dyson, Jennifer L. Beebe-Dimmer, Lifang Hou, Barbara V. Howard, Ross Prentice, Michael S. Simon. The effect of genetic variants on the relationship between statins and breast cancer in the Women's Health Initiative Observational Study. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 07.

Yersinia enterocolitica YopT and Clostridium difficile Toxin B Induce Expression of GILZ in Epithelial Cells

Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype Yersinia enterocolitica which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different Yersinia mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, Clostridium difficile toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal GILZ promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the GILZ promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of GILZ promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.

Protocadherin clusters and cell adhesion kinase regulate dendrite complexity through Rho GTPase

Dendritic patterning and spine morphogenesis are crucial for the assembly of neuronal circuitry to ensure normal brain development and synaptic connectivity as well as for understanding underlying mechanisms of neuropsychiatric diseases and cognitive impairments. The Rho GTPase family is essential for neuronal morphogenesis and synaptic plasticity by modulating and reorganizing the cytoskeleton. Here, we report that protocadherin (Pcdh) clusters and cell adhesion kinases (CAKs) play important roles in dendritic development and spine elaboration. The knockout of the entire Pcdhα cluster results in the dendritic simplification and spine loss in CA1 pyramidal neurons in vivo and in cultured primary hippocampal neurons in vitro. The knockdown of the whole Pcdhγ cluster or in combination with the Pcdhα knockout results in similar dendritic and spine defects in vitro. The overexpression of proline-rich tyrosine kinase 2 (Pyk2, also known as CAKβ, RAFTK, FAK2, and CADTK) recapitulates these defects and its knockdown rescues the phenotype. Moreover, the genetic deletion of the Pcdhα cluster results in phosphorylation and activation of Pyk2 and focal adhesion kinase (Fak) and the inhibition of Rho GTPases in vivo. Finally, the overexpression of Pyk2 leads to inactivation of Rac1 and, conversely, the constitutive active Rac1 rescues the dendritic and spine morphogenesis defects caused by the knockout of the Pcdhα cluster and the knockdown of the Pcdhγ cluster. Thus, the involvement of the Pcdh-CAK-Rho GTPase pathway in the dendritic development and spine morphogenesis has interesting implications for proper assembly of neuronal connections in the brain.

Tumor Necrosis Factor α Decreases Nitric Oxide Synthase Type 3 Expression Primarily via Rho/Rho Kinase in the Thick Ascending Limb

Inappropriate Na(+) reabsorption by thick ascending limbs (THALs) induces hypertension. NO produced by NO synthase type 3 (NOS3) inhibits NaCl reabsorption by THALs. Tumor necrosis factor α (TNF-α) decreases NOS3 expression in endothelial cells and contributes to increases in blood pressure. However, the effects of TNF-α on THAL NOS3 and the signaling cascade are unknown. TNF-α activates several signaling pathways, including Rho/Rho kinase (ROCK), which is known to reduce NOS3 expression in endothelial cells. Therefore, we hypothesized that TNF-α decreases NOS3 expression via Rho/ROCK in rat THAL primary cultures. THAL cells were incubated with either vehicle or 1 nmol/L of TNF-α for 24 hours, and NOS3 expression was measured by Western blot. TNF-α decreased NOS3 expression by 51 ± 6% (P<0.002) and blunted stimulus-induced NO production. A 10-minute treatment with TNF-α stimulated RhoA activity by 60 ± 23% (P<0.04). Inhibition of Rho GTPase with 0.05 μg/mL of C3 exoenzyme blocked TNF-α-induced reductions in NOS3 expression by 30 ± 8% (P<0.02). Inhibition of ROCK with 10 μmol/L of H-1152 blocked TNF-α-induced decreases in NOS3 expression by 66 ± 15% (P<0.001). Simultaneous inhibition of Rho and ROCK had no additive effect. Myosin light chain kinase, NO, protein kinase C, mitogen-activated kinase kinase, c-Jun amino terminal kinases, and Rac-1 were also not involved in TNF-α-induced decreases in NOS3 expression. We conclude that TNF-α decreases NOS3 expression primarily via Rho/ROCK in rat THALs. These data suggest that some of the beneficial effects of ROCK inhibitors in hypertension could be attributed to the mitigation of TNF-α-induced reduction in NOS3 expression.

Abstract 32: P190B RhoGAP overexpression drives mammary epithelial cell proliferation by altering stromal-epithelial interactions in the developing mammary gland

Abstract Breast cancer development and progression is enabled by alterations in the surrounding stroma originating from stromal interactions with cancerous epithelium. Rho GTPases and their regulators have been implicated as important mediators of stromal-epithelial interactions in mammary epithelial cells (MECs), and alterations in the microenvironment profoundly affect MEC morphogenesis in a Rho GTPase-dependent manner. Previously, we demonstrated that p190B Rho GTPase activating protein (RhoGAP), a major inhibitor of Rho GTPases, has pro-tumorigenic functions during MMTV-Neu induced mammary tumorigenesis. We hypothesized that p190B overexpression in the epithelium promotes mammary tumor development by altering stromal-epithelial interactions to drive MEC proliferation and disrupt tissue architecture. To investigate this hypothesis, we examined the effects of p190B overexpression in the developing mammary gland (MG) using Tet-regulatable p190B overexpressing mice. Interestingly, overexpression of p190B in the mammary epithelium during postnatal MG development causes hyperbranching and disorganization of the ductal tree. Using a combination of in vivo morphometric and immunohistochemical analyses and in vitro stromal-epithelial co-cultures, we have demonstrated that p190B overexpression in MECs activates fibroblasts in the adjacent microenvironment leading to increased extracellular matrix (ECM) deposition, elevated expression of the collagen crosslinking enzyme lysyl oxidase, and increased expression of potent regulators of branching morphogenesis, including FGF and TGFβ growth factors. These stromal alterations correlated with increased proliferation of p190B overexpressing MECs in vivo. Taken together, these data suggest that p190B overexpression may contribute to mammary tumorigenesis by creating a pro-tumorigenic stromal environment leading to increased MEC proliferation and altered tissue architecture. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 32. doi:1538-7445.AM2012-32

Targeting Rho GTPase Signaling for Cancer Therapy

Accumulating evidence from basic and clinical studies supports the concept that signaling pathways downstream of Rho GTPases play important roles in tumor development and progression. As a result, there has been considerable interest in the possibility that specific proteins in these signal transduction pathways could be potential targets for cancer therapy. A number of inhibitors targeting critical effector proteins, activators or the Rho GTPases themselves, have been developed. We will review the strategies currently being used to develop inhibitors of Rho GTPases and downstream signaling kinases and discuss candidate entities. Although molecularly targeted drugs that inhibit Rho GTPase signaling have not yet been widely adopted for clinical use, their potential value as cancer therapeutics continues to drive considerable pharmaceutical research and development.