Rac1 Function Research Articles

Effects of RAC1 on Proliferation of Hen Ovarian Prehierarchical Follicle Granulosa Cells.

Simple SummaryThe growth and development of ovary follicles is an intricate, highly organized process involving many local intra-ovarian factors. Ras-related C3 botulinum toxin substrate1 (RAC1) is speculated to be associated with prehierarchical follicle development of hen ovaries. The current study initially revealed RAC1 mRNA to be expressed in varied-size follicles and stroma and its expression levels in the prehierarchical follicles of 1.0–3.9 mm, 6.0–6.9 mm and 7.0–8.0 mm in diameter were remarkably higher than the other groups. Moreover, RAC1 protein was mainly expressed in the oocytes and granulosa cells (GC), as well as in stromal tissues of the follicles. To understand the exact roles of the RAC1 gene in regulation of follicular GC proliferation and differentiation, siRNA interference and overexpression of the RAC1 gene were conducted. Our experiments demonstrated that the RAC1 gene can significantly promote the expression of mRNA and proteins of FSHR, CCND2, CYP11A1, PCNA and StAR genes in GC and directly elevate the proliferation of GC in vitro. These results indicated RAC1 played a crucial role in regulation of GC proliferation and differentiation and steroidogenesis during the development of prehierarchical follicles. This study provided a base for elucidating the molecular mechanisms underlying the biological effect of RAC1 on the hen ovary follicle growth and development.RAC1 belongs to the small G protein Rho subfamily and is implicated in regulating gene expression, cell proliferation and differentiation in mammals and humans; nevertheless, the function of RAC1 in growth and development of hen ovarian follicles is still unclear. This study sought to understand the biological effects of RAC1 on granulosa cell (GC) proliferation and differentiation of hen ovarian prehierarchical follicles. Firstly, our results showed expression levels of RAC1 mRNA in the follicles with diameters of 7.0–8.0 mm, 6.0–6.9 mm and 1.0–3.9 mm were greater than other follicles (p < 0.05). The RAC1 protein was mainly expressed in oocyte and its around GCs and stromal tissues of the prehierarchical follicles by immunohistochemistry. Further investigation revealed the RAC1 gene remarkably enhanced the mRNA and protein expression levels of FSHR (a marker of follicle selection), CCND2 (a marker of cell-cycle progression and GC differentiation), PCNA (a marker of GC proliferation), StAR and CYP11A1 (markers of GC differentiation and steroidogenesis) (p < 0.05). Furthermore, our data demonstrated siRNA interference of RAC1 significantly reduced GC proliferation (p < 0.05), while RAC1 gene overexpression enhanced GC proliferation in vitro (p < 0.05). Collectively, this study provided new evidence that the biological effects of RAC1 on GC proliferation, differentiation and steroidogenesis of chicken ovary follicles.

A PI3K- and GTPase-independent Rac1-mTOR mechanism mediates MET-driven anchorage-independent cell growth but not migration.

Receptor tyrosine kinases (RTKs) are often overexpressed or mutated in cancers and drive tumor growth and metastasis. In the current model of RTK signaling, including that of MET, downstream phosphatidylinositol 3-kinase (PI3K) mediates both cell proliferation and cell migration, whereas the small guanosine triphosphatase (GTPase) Rac1 mediates cell migration. However, in cultured NIH3T3 and glioblastoma cells, we found that class I PI3K mediated oncogenic MET-induced cell migration but not anchorage-independent growth. In contrast, Rac1 regulated both processes in distinct ways. Downstream of PI3K, Rac1 mediated cell migration through its GTPase activity, whereas independently of PI3K, Rac1 mediated anchorage-independent growth in a GTPase-independent manner through an adaptor function. Through its RKR motif, Rac1 formed a complex with the kinase mTOR to promote its translocation to the plasma membrane, where its activity promoted anchorage-independent growth of the cell cultures. Inhibiting mTOR with rapamycin suppressed the growth of subcutaneous MET-mutant cell grafts in mice, including that of MET inhibitor-resistant cells. These findings reveal a GTPase-independent role for Rac1 in mediating a PI3K-independent MET-to-mTOR pathway and suggest alternative or combined strategies that might overcome resistance to RTK inhibitors in patients with cancer.

Active RAC1 Promotes Tumorigenic Phenotypes and Therapy Resistance in Solid Tumors.

Acting as molecular switches, all three members of the Guanosine triphosphate (GTP)-ase-family, Ras-related C3 botulinum toxin substrate (RAC), Rho, and Cdc42 contribute to various processes of oncogenic transformations in several solid tumors. We have reviewed the distribution of patterns regarding the frequency of Ras-related C3 botulinum toxin substrate 1 (RAC1)-alteration(s) and their modes of actions in various cancers. The RAC1 hyperactivation/copy-number gain is one of the frequently observed features in various solid tumors. We argued that RAC1 plays a critical role in the progression of tumors and the development of resistance to various therapeutic modalities applied in the clinic. With this perspective, here we interrogated multiple functions of RAC1 in solid tumors pertaining to the progression of tumors and the development of resistance with a special emphasis on different tumor cell phenotypes, including the inhibition of apoptosis and increase in the proliferation, epithelial-to-mesenchymal transition (EMT), stemness, pro-angiogenic, and metastatic phenotypes. Our review focuses on the role of RAC1 in adult solid-tumors and summarizes the contextual mechanisms of RAC1 involvement in the development of resistance to cancer therapies.

Distinct Roles for Rac1 in Sertoli Cell Function during Testicular Development and Spermatogenesis

SUMMARYSertoli cells are supporting cells of the testicular seminiferous tubules, which provide a nurturing environment for spermatogenesis. Adult Sertoli cells are polarized so that they can simultaneously support earlier-stage spermatogenic cells (e.g., spermatogonia) basally and later-stage cells (e.g., spermatids) apically. To test the consequences of disrupting cell polarity in Sertoli cells, we perform a Sertoli-specific conditional deletion of Rac1, which encodes a Rho GTPase required for apicobasal cell polarity. Rac1 conditional knockout adults exhibit spermatogenic arrest at the round spermatid stage, with severe disruption of Sertoli cell polarity, and show increased germline and Sertoli cell apoptosis. Thus, Sertoli Rac1 function is critical for the progression of spermatogenesis but, surprisingly, is dispensable for fetal testicular development, adult maintenance of undifferentiated spermatogonia, and meiotic entry. Our data indicate that Sertoli Rac1 function is required only for certain aspects of spermatogenesis and reveal that there are distinct requirements for cell polarity during cellular differentiation.

ATP synthase inhibitory factor 1 (IF1), a novel myokine, regulates glucose metabolism by AMPK and Akt dual pathways.

ATPase inhibitory factor 1 (IF1) is an ATP synthase-interacting protein that suppresses the hydrolysis activity of ATP synthase. In this study, we observed that the expression of IF1 was up-regulated in response to electrical pulse stimulation of skeletal muscle cells and in exercized mice and healthy men. IF1 stimulates glucose uptake via AMPK in skeletal muscle cells and primary cultured myoblasts. Reactive oxygen species and Rac family small GTPase 1 (Rac1) function in the upstream and downstream of AMPK, respectively, in IF1-mediated glucose uptake. In diabetic animal models, the administration of recombinant IF1 improved glucose tolerance and down-regulated blood glucose level. In addition, IF1 inhibits ATP hydrolysis by β-F1-ATPase in plasma membrane, thereby increasing extracellular ATP and activating the protein kinase B (Akt) pathway, ultimately leading to glucose uptake. Thus, we suggest that IF1 is a novel myokine and propose a mechanism by which AMPK and Akt contribute independently to IF1-mediated improvement of glucose tolerance impairment. These results demonstrate the importance of IF1 as a potential antidiabetic agent.-Lee, H. J., Moon, J., Chung, I., Chung, J. H., Park, C., Lee, J. O., Han, J. A., Kang, M. J., Yoo, E. H., Kwak, S.-Y., Jo, G., Park, W., Park, J., Kim, K. M., Lim, S., Ngoei, K. R. W., Ling, N. X. Y., Oakhill, J. S., Galic, S., Murray-Segal, L., Kemp, B. E., Mantzoros, C. S., Krauss, R. M., Shin, M.-J., Kim, H. S. ATP synthase inhibitory factor 1 (IF1), a novel myokine, regulates glucose metabolism by AMPK and Akt dual pathways.

The Rac3 GTPase in Neuronal Development, Neurodevelopmental Disorders, and Cancer.

Rho family small guanosine triphosphatases (GTPases) are important regulators of the cytoskeleton, and are critical in many aspects of cellular and developmental biology, as well as in pathological processes such as intellectual disability and cancer. Of the three members of the family, Rac3 has a more restricted expression in normal tissues compared to the ubiquitous member of the family, Rac1. The Rac3 polypeptide is highly similar to Rac1, and orthologues of the gene for Rac3 have been found only in vertebrates, indicating the late appearance of this gene during evolution. Increasing evidence over the past few years indicates that Rac3 plays an important role in neuronal development and in tumor progression, with specificities that distinguish the functions of Rac3 from the established functions of Rac1 in these processes. Here, results highlighting the importance of Rac3 in distinct aspects of neuronal development and tumor cell biology are presented, in support of the non-redundant role of different members of the two Rac GTPases in physiological and pathological processes.

RAC1 Takes the Lead in Solid Tumors.

Three GTPases, RAC, RHO, and Cdc42, play essential roles in coordinating many cellular functions during embryonic development, both in healthy cells and in disease conditions like cancers. We have presented patterns of distribution of the frequency of RAC1-alteration(s) in cancers as obtained from cBioPortal. With this background data, we have interrogated the various functions of RAC1 in tumors, including proliferation, metastasis-associated phenotypes, and drug-resistance with a special emphasis on solid tumors in adults. We have reviewed the activation and regulation of RAC1 functions on the basis of its sub-cellular localization in tumor cells. Our review focuses on the role of RAC1 in cancers and summarizes the regulatory mechanisms, inhibitory efficacy, and the anticancer potential of RAC1-PAK targeting agents.

Fungal homologues of human Rac1 as emerging players in signal transduction and morphogenesis.

A wealth of data is accumulating on the physiological functions of human Rac1, a member of the Rho GTPase family of molecular switches and substrate of botulinum toxin, which was first identified as a regulator of cell motility through its effect on the actin cytoskeleton. Later on, it was found to be involved in different diseases like cancers, cardiac function, neuronal disorders, and apoptotic cell death. Despite the presence of Rac1 homologues in most fungi investigated so far, including Rho5 in the genetically tractable model yeast Saccharomyces cerevisiae, knowledge on their physiological functions is still scarce, let alone the details of the molecular mechanisms of their actions and interactions. Nevertheless, all functions proposed for human Rac1 seem to be conserved in one or the other fungus. This includes the regulation of MAPK cascades, polarized growth, and actin dynamics. Moreover, both the production and response to reactive oxygen species, as well as the reaction to nutrient availability, can be affected. We here summarize the studies performed on fungal Rac1 homologues, with a special focus on S. cerevisiae Rho5, which may be of use in drug development in medicine and agriculture.

BMP7 coordinates endometrial epithelial cell receptivity for blastocyst implantation through the endoglin pathway in cell lines and a mouse model.

Bone morphogenetic protein (BMP) expression has been observed in the uterus in previous studies. However, the influence of BMP7 on blastocyst implantation remains unclear. Blastocysts first act on luminal endometrial epithelial cells during implantation. The purpose of the present study was to explore the influence of BMP7 on endometrial epithelial cells. A pregnancy animal model, and mouse and human endometrial epithelial cells were used in the present study. Transient knockdown, immunofluorescence assay, in vitro embryo implantation, BMP7 silencing, reverse transcription-quantitative polymerase chain reaction, western blotting, immunoprecipitation and Rac1 function assay were also performed. It was revealed that BMP7 concentration was increased in endometrial epithelial cells during the final pre-receptive and receptive stages of receptivity in the mouse endometrium. Additionally, BM7 acted on the transforming growth factor-β receptor, endoglin. Endoglin expression was detected in both stromal and endothelial cells apart from trophoblast expression. Following knockdown of BMP7, Rac-GTP was decreased in endometrial epithelial cells and the uterus. Knockdown of endoglin by small interfering RNA decreased the number of blastocysts and implantation regions. Additionally, BMP7 silencing and endoglin suppression of Ishikawa cells led to impaired JAr spheroid attachment. These findings suggest that BMP7 is associated with receptivity of the endometrium, indicating that BMP7 regulates receptivity of endometrial epithelial cells for implantation of blastocysts via the endoglin pathway.

Post-Translational Modification and Subcellular Distribution of Rac1: An Update.

Rac1 is a small GTPase that belongs to the Rho family. The Rho family of small GTPases is a subfamily of the Ras superfamily. The Rho family of GTPases mediate a plethora of cellular effects, including regulation of cytoarchitecture, cell size, cell adhesion, cell polarity, cell motility, proliferation, apoptosis/survival, and membrane trafficking. The cycling of Rac1 between the GTP (guanosine triphosphate)- and GDP (guanosine diphosphate)-bound states is essential for effective signal flow to elicit downstream biological functions. The cycle between inactive and active forms is controlled by three classes of regulatory proteins: Guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine-nucleotide-dissociation inhibitors (GDIs). Other modifications include RNA splicing and microRNAs; various post-translational modifications have also been shown to regulate the activity and function of Rac1. The reported post-translational modifications include lipidation, ubiquitination, phosphorylation, and adenylylation, which have all been shown to play important roles in the regulation of Rac1 and other Rho GTPases. Moreover, the Rac1 activity and function are regulated by its subcellular distribution and translocation. This review focused on the most recent progress in Rac1 research, especially in the area of post-translational modification and subcellular distribution and translocation.

Impact of Charge Transport Dynamics and Conditioning on Cycling Efficiency within Single Redox Active Colloids

AbstractRedox active colloids (RACs) are flowable suspensions for size‐exclusion redox flow batteries that exchange billions of electrons per particle. Single particle measurements via bulk electrolysis and voltammetry provided an accelerated platform for evaluating the role of state of charge and conditioning on RAC performance. We used scanning electrochemical microscopy to image, isolate, and interrogate RACs (830 nm diameter) with a 300 nm probe. Deep electrolysis of the particles evidenced capacity losses, but this conditioning simultaneously led to increased Coulombic efficiency. On the other hand, shallow cycling using voltammetry for over 150 cycles showed improved charge recovery and gradual changes in the particle's diffusional regimes. Raman spectroelectrochemistry on few RACs confirmed that degradation occurred upon deep cycling but that shallow cycling was not as detrimental, albeit with low capacity access. The methodologies presented herein provide a stepwise viewpoint of the progression of RAC function with cycling, linking bulk behavior with that of individual particles.

An Intellectual Disability-Related Missense Mutation in Rac1 Prevents LTP Induction.

The small GTPase Rac1 promotes actin polymerization and plays a critical and increasingly appreciated role in the development and plasticity of glutamatergic synapses. Growing evidence suggests that disruption of the Rac1 signaling pathway at glutamatergic synapses contributes to Autism Spectrum Disorder/intellectual disability (ASD/ID)-related behaviors seen in animal models of ASD/ID. Rac1 has also been proposed as a strong candidate of convergence for many factors implicated in the development of ASD/ID. However, the effects of ASD/ID-related mutations in Rac1 itself have not been explored in neurons. Here, we investigate a recently reported de novo missense mutation in Rac1 found in an individual with severe ID. Our modeling predicts that this mutation will strongly inhibit Rac1 activation by occluding Rac1’s GTP binding pocket. Indeed, we find that this de novo mutation prevents Rac1 function and results in a selective reduction in synaptic AMPA receptor function. Furthermore, this mutation prevents the induction of long-term potentiation (LTP), the cellular mechanism underlying learning and memory formation. Together, our findings strongly suggest that this mutation contributes to the development of ID in this individual. This research demonstrates the importance of Rac1 in synaptic function and plasticity and contributes to a growing body of evidence pointing to dysregulation of actin polymerization at glutamatergic synapses as a contributing factor to ASD/ID.

Cooperation of Rho family proteins Rac1 and Cdc42 in cartilage development and calcified tissue formation

Rac1 and Cdc42, Rho family low molecular weight G proteins, are intracellular signaling factors that transmit various information from outside to inside cells. Primarily, they are known to control various biological activities mediated by actin cytoskeleton reorganization, such as cell proliferation, differentiation, and apoptosis. In order to investigate the functions of Rac1 and Cdc42 in bone formation, we prepared cartilage-specific double conditional knockout mice, Rac1fl/fl; Cdc42fl/fl; Col2-Cre (Rac1: Cdc42 dcKO mice), which died just after birth, similar to Cdc42fl/fl; Col2-Cre mice (Cdc42 cKO mice). Our findings showed that the long tubule bone in Rac1: Cdc42 dcKO mice was shorter than that in Rac1fl/fl; Col2-Cre mice (Rac1 cKO mice) and Cdc42 cKO mice. Abnormal skeleton formation was also observed and disordered columnar formation in the growth plate of the Rac1: Cdc42 dcKO mice was more severe as compared to the Rac1 cKO and Cdc42 cKO mice. Together, these results suggest that Rac1 and Cdc42 have cooperating roles in regulation of bone development.

The Small GTPase RAC1/CED-10 Is Essential in Maintaining Dopaminergic Neuron Function and Survival Against \u03b1-Synuclein-Induced Toxicity

Parkinson’s disease is associated with intracellular α-synuclein accumulation and ventral midbrain dopaminergic neuronal death in the Substantia Nigra of brain patients. The Rho GTPase pathway, mainly linking surface receptors to the organization of the actin and microtubule cytoskeletons, has been suggested to participate to Parkinson’s disease pathogenesis. Nevertheless, its exact contribution remains obscure. To unveil the participation of the Rho GTPase family to the molecular pathogenesis of Parkinson’s disease, we first used C elegans to demonstrate the role of the small GTPase RAC1 (ced-10 in the worm) in maintaining dopaminergic function and survival in the presence of alpha-synuclein. In addition, ced-10 mutant worms determined an increase of alpha-synuclein inclusions in comparison to control worms as well as an increase in autophagic vesicles. We then used a human neuroblastoma cells (M17) stably over-expressing alpha-synuclein and found that RAC1 function decreased the amount of amyloidogenic alpha-synuclein. Further, by using dopaminergic neurons derived from patients of familial LRRK2-Parkinson’s disease we report that human RAC1 activity is essential in the regulation of dopaminergic cell death, alpha-synuclein accumulation, participates in neurite arborization and modulates autophagy. Thus, we determined for the first time that RAC1/ced-10 participates in Parkinson’s disease associated pathogenesis and established RAC1/ced-10 as a new candidate for further investigation of Parkinson’s disease associated mechanisms, mainly focused on dopaminergic function and survival against α-synuclein-induced toxicity.

Roles of Cdc42 and Rac in Bergmann glia during cerebellar corticogenesis

Bergmann glia (BG) are important in the inward type of radial migration of cerebellar granule neurons (CGNs). However, details regarding the functions of Cdc42 and Rac in BG for radial migration of CGN are unknown. To examine the roles of Cdc42 and Rac in BG during cerebellar corticogenesis, mice with a single deletion of Cdc42 or Rac1 and those with double deletions of Cdc42 and Rac1 under control of the glial fibrillary acidic protein (GFAP) promoter: GFAP-Cre;Cdc42flox/flox (Cdc42-KO), GFAP–Cre;Rac1flox/flox (Rac1-KO), and GFAP-Cre; Cdc42flox/flox;Rac1flox/flox (Cdc42/Rac1-DKO) mice, were generated. Both Cdc42-KO and Rac1-KO mice, but more obviously Cdc42-KO mice, had disturbed alignment of BG in the Purkinje cell layer (PCL). We found that Cdc42-KO, but not Rac1-KO, induced impaired radial migration of CGNs in the late phase of radial migration, leading to retention of CGNs in the lower half of the molecular layer (ML). Cdc42-KO, but not Rac1-KO, mice also showed aberrantly aligned Purkinje cells (PCs). These phenotypes were exacerbated in Cdc42/Rac1-DKO mice. Alignment of BG radial fibers in the ML and BG endfeet at the pial surface of the cerebellum evaluated by GFAP staining was disturbed and weak in Cdc42/Rac1-DKO mice, respectively. Our data indicate that Cdc42 and Rac, but predominantly Cdc42, in BG play important roles during the late phase of radial migration of CGNs. We also report here that Cdc42 is involved in gliophilic migration of CGNs, in contrast to Rac, which is more closely connected to regulating neurophilic migration.

The phosphomimetic mutation of syndecan-4 binds and inhibits Tiam1 modulating Rac1 activity in PDZ interaction-dependent manner.

The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1–RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways.

TGF-β1-induced cell migration in pancreatic carcinoma cells is RAC1 and NOX4-dependent and requires RAC1 and NOX4-dependent activation of p38MAPK.

Transforming growth factor (TGF)-β promotes epithelial-mesenchymal transition and cell invasion of cancer cells in part through the small GTPase RAC1. Since RAC1 can signal through reactive oxygen species (ROS), we probed the role of the ROS-producing NADPH oxidase (NOX) and p38 mitogen-activated protein kinase (MAPK) in mediating TGF-β1/RAC1-driven random cell migration (chemokinesis). Although the NOX isoforms NOX2, 4, 5,6, and RAC1 were readily detectable by RT-PCR in pancreatic ductal adenocarcinoma (PDAC)-derived Panc1 and Colo357 cells, only NOX4 and RAC1 were expressed at higher levels comparable to those in peripheral blood monocytes. TGF-β1 treatment resulted in upregulation of NOX4 (and NOX2) and rapid intracellular production of ROS. To analyze whether RAC1 functions through NOX and ROS to promote cell motility, we performed real-time cell migration assays with xCELLigence® technology in the presence of the ROS scavenger N-acetyl-L-cysteine (NAC) and various NOX inhibitors. NAC, the NOX4 inhibitor diphenylene iodonium or small interfering RNA (siRNA) to NOX4, and the NOX2 inhibitor apocynin all suppressed TGF-β1-induced chemokinesis of Panc1 and Colo357 cells as did various inhibitors of RAC1 used as control. In addition, we showed that blocking NOX4 or RAC1 function abrogated phosphorylation of p38MAPK signaling by TGF-β1 and that inhibition of p38MAPK reduced TGF-β1-induced random cell migration, while ectopic expression of a kinase-active version of the p38 activating kinase MKK6 was able to partially rescue the decline in migration after RAC1 inhibition. Our data suggest that TGF-β1-induced chemokinesis in PDAC cells is mediated through a RAC1/NOX4/ROS/p38MAPK cascade.

CDNA cloning, expression and immune function analysis of a novel Rac1 gene (AjRac1) in the sea cucumber Apostichopus japonicus

The ras-related C3 botulinum toxin substrate 1 (Rac1) belongs to Ras homolog (Rho) small GTPases subfamily. As an important molecular switch, Rac1 regulates various processes in the cell, especially in cellular immune response. With attempt to clarify characters and functions of Rac1 in sea cucumbers, full length cDNA of a Rac1 homolog in the sea cucumber Apostichopus japonicus (AjRac1) was cloned by transcriptome database mining and rapid amplification of cDNA ends (RACE) techniques. The open reading frame of AjRac1 is 579 bp encoding a protein with a length of 192 aa. Sequence analysis showed that AjRac1 is highly conserved as compared to those from other eukaryotic species. Phylogenetic analysis revealed that amino acid sequence of AjRac1 closely related to those from Strongylocentrotus purpuratus. Results of expression analysis showed that AjRac1 exhibited a relative high expression in blastula stage, adult coelomocytes and respiratory tree in A. japonicus. The transcription of AjRac1 in adult coelomocytes altered significantly at 4 h- and 12 h-after Vibrio splendidus infection, respectively, which indicated that AjRac1 involved in sea cucumber innate immunity. All data presented in this study will deepen our understanding of characterizations and immunological functions of Rac1 in sea cucumbers.

Increased Training Intensity Induces Proper Membrane Localization of Actin Remodeling Proteins in the Hippocampus Preventing Cognitive Deficits: Implications for Fragile X Syndrome.

Behavioral intervention therapy has proven beneficial in the treatment of autism and intellectual disabilities (ID), raising the possibility of certain changes in molecular mechanisms activated by these interventions that may promote learning. Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by autistic features and intellectual disability and can serve as a model to examine mechanisms that promote learning. FXS results from mutations in the fragile X mental retardation 1 gene (Fmr1) that prevents expression of the Fmr1 protein (FMRP), a messenger RNA (mRNA) translation regulator at synapses. Among many other functions, FMRP organizes a complex with the actin cytoskeleton-regulating small Rho GTPase Rac1. As in humans, Fmr1 KO mice lacking FMRP display autistic-like behaviors and deformities of actin-rich synaptic structures in addition to impaired hippocampal learning and synaptic plasticity. These features have been previously linked to proper function of actin remodeling proteins that includes Rac1. An important step in Rac1 activation and function is its translocation to the membrane, where it can influence synaptic actin cytoskeleton remodeling during hippocampus-dependent learning. Herein, we report that Fmr1 KO mouse hippocampus exhibits increased levels of membrane-bound Rac1, which may prevent proper learning-induced synaptic changes. We also determine that increasing training intensity during fear conditioning (FC) training restores contextual memory in Fmr1 KO mice and reduces membrane-bound Rac1 in Fmr1 KO hippocampus. Increased training intensity also results in normalized long-term potentiation in hippocampal slices taken from Fmr1 KO mice. These results point to interventional treatments providing new therapeutic options for FXS-related cognitive dysfunction.

Abstract MIP-061: RAC 1 AND CDC42 GTPASES AS REGULATORS OF OVARIAN CANCER PROGRESSION AND NICHE INTERACTIONS

Abstract PURPOSE OF THE STUDY: The Ras-homologous (Rho) family GTPases Rac1 and Cdc42 contribute to metastatic dissemination through regulation of actin reorganization, cell motility, cell-cell and cell-extracellular matrix adhesion. The purpose of the study was to investigate the contributions of these GTPases in ovarian cancer progression. EXPERIMENTAL PROCEDURES: GTPase target expression and activity was determined by analysis of The Cancer Genome Atlas (TCGA), immunohistochemistry of ovarian cancer tissues and enzymatic assays of patient samples. The effects Rac1 and Cdc42 expression were investigated by comparing cell behaviors in vitro and in vivo in engineered cell lines and use of inhibitors of Rac1, Cdc42 or dual inhibition by the R-enantiomer of ketorolac. SUMMARY OF THE DATA: Expression of the constitutively active Rac1 splice variant Rac1b and elevation of Cdc42 protein were detected in advanced ovarian cancer specimens thus providing the first evidence for dysregulation of these GTPase targets in ovarian cancer. To confirm the importance of Rac1, RNA-seq gene expression data from 305 Stage III or IV ovarian cancer samples were downloaded from The Cancer Genome Atlas (TCGA) and evaluated for RAC1 gene expression. The samples with the highest Rac1 RNA levels had significantly worse (p=0.039) age adjusted survival than the patients with the lowest Rac1 levels. Additional analyses identified more than 4,000 genes that were differentially expressed (FDR corrected p &amp;lt;0.05) between these patient groups. Gene Ontology analysis revealed several highly enriched biological processes including: extracellular matrix organization (67 genes p=1.60E-25), multicellular organismal catabolic processes (29 genes p=4.30E-22), regulation of epithelial cell proliferation (30 genes p=6.10E-09 and regulation of cell migration (48 genes p=1.50E-08). Relevance of the pathway analyses is illustrated by experimental evidence that increased GTPase expression leads to enhanced homing and engraftment in the omental niche; suggesting that elevated activity detected in patients may contribute to disease progression. Conversely, dual inhibition of Rac1 and Cdc42 activity by R-ketorolac inhibits ovarian patient-derived tumor cell adhesion and invasion, omental homing and tumor implantation in a mouse xenograft model. CONCLUSIONS: These findings indicate that high expression of Rac1 is associated with late stage disease and/or poor patient survival and with genes involved in extracellular matrix, cell proliferation and cell migration. These functions of Rac1 were confirmed in experimental models and inhibition of protein activity provided anti-tumor benefit in vivo. Together, these data suggest that Rac1 and/or Cdc42 function as drivers of ovarian cancer and may represent novel therapeutic targets to improve ovarian cancer outcome. Citation Format: Hudson LG, Kenney SR, Rivera, M., Weisend, J. Luo, L., Ness, S., Gillette, JM, Wandinger-Ness A. RAC 1 AND CDC42 GTPASES AS REGULATORS OF OVARIAN CANCER PROGRESSION AND NICHE INTERACTIONS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-061.