Differential expression of Rac GTPase activating protein 1 in triple negative breast cancer.

Women diagnosed with triple negative breast cancer are predicted to benefit neither from endocrine therapy nor from HER2-targeted therapies (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding Rac GTPase activating protein 1, RACGAP1, when comparing the tumor cells of patients with triple negative breast cancer to normal mammary ductal cells (2). RACGAP1 was also differentially expressed in bulk tumor in human breast cancer (3). RACGAP1 mRNA was present at significantly increased quantities in TNBC tumor cells relative to normal mammary ductal cells. Analysis of human survival data revealed that expression of RACGAP1 in primary tumors of the breast was correlated with overall survival in patients with luminal A and HER2+ type cancer, while within triple negative breast cancer, primary tumor expression of RACGAP1 was correlated with distant metastasis-free survival in patients with immunomodulatory, mesenchymal stem-like, and luminal androgen receptor subtype disease. RACGAP1 may be of relevance to initiation, maintenance or progression of triple negative breast cancers.

Rac GTPase Signaling in Immune-Mediated Mechanisms of Atherosclerosis

Coronary artery disease caused by atherosclerosis is a major cause of morbidity and mortality around the world. Data from preclinical and clinical studies support the belief that atherosclerosis is an inflammatory disease that is mediated by innate and adaptive immune signaling mechanisms. This review sought to highlight the role of Rac-mediated inflammatory signaling in the mechanisms driving atherosclerotic calcification. In addition, current clinical treatment strategies that are related to targeting hypercholesterolemia as a critical risk factor for atherosclerotic vascular disease are addressed in relation to the effects on Rac immune signaling and the implications for the future of targeting immune responses in the treatment of calcific atherosclerosis.

Over-expression of Rac GTPase activating protein 1 in human endometrial cancer.

Gynecologic cancers including cancers of the endometrium are a clinical problem (1-4). We mined published microarray data (5, 6) to discover genes associated with endometrial cancers by comparing transcriptomes of the normal endometrium and endometrial tumors from humans. We identified Rac GTPase activating protein 1, encoded by RACGAP1, as among the most differentially expressed genes, transcriptome-wide, in cancers of the endometrium. RACGAP1 was expressed at significantly higher levels in endometrial tumor tissues as compared to the endometrium. Importantly, primary tumor expression of RACGAP1 was correlated with overall survival in patients with endometrial cancer. RACGAP1 may be a molecule of interest in understanding the etiology or progression of human endometrial cancer.

Role of PIKfyve in macrophage and neutrophil immune response

Solid particles such as pathogens, dying cells, and debris are engulfed by macrophages and neutrophils and sequestered into a phagosome. Phagosomes fuse with early and late endosomes and ultimately with lysosomes to mature into phagolysosomes, a process known as phagosome maturation. The formation of highly acidic and degradative phagolysosomes plays an important role in degradation of the internalized particle. We employed siRNA and pharmacological tools to demonstrate that phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2], synthesized by the PIKfyve lipid kinase, is required for phagosome maturation. However, the mechanism by which PI(3,5)P2 controls phagosome maturation remained uncharacterized. We hypothesized that PI(3,5)P2 may control phagosome-lysosome fusion partly by stimulating TRPML1, a lysosomal Ca2+ channel gated by PI(3,5)P2. Upon opening of the channel, lysosomal Ca2+ would diffuse and trigger phagosome-lysosome fusion since Ca2+ is known to induce membrane fusion post-docking of SNARE proteins. In addition, we also demonstrated that the lipid kinase PIKfyve coordinates the neutrophils immune response by controlling phagosome maturation and regulating Rac GTPase activity. PIKfyve produces both PI(3,5)P2 and phosphatidylinositol-5-phosphate (PI5P); therefore, it might control phagosome maturation through production of PI(3,5)P2 and activation of TRPML1 as well as regulates ROS production and chemotaxis through synthesis of PI5P, which leads to the activation of Tiam1, and Rac GTPase.

Role of PIKfyve in macrophage and neutrophil immune response

Solid particles such as pathogens, dying cells, and debris are engulfed by macrophages and neutrophils and sequestered into a phagosome. Phagosomes fuse with early and late endosomes and ultimately with lysosomes to mature into phagolysosomes, a process known as phagosome maturation. The formation of highly acidic and degradative phagolysosomes plays an important role in degradation of the internalized particle. We employed siRNA and pharmacological tools to demonstrate that phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2], synthesized by the PIKfyve lipid kinase, is required for phagosome maturation. However, the mechanism by which PI(3,5)P2 controls phagosome maturation remained uncharacterized. We hypothesized that PI(3,5)P2 may control phagosome-lysosome fusion partly by stimulating TRPML1, a lysosomal Ca2+ channel gated by PI(3,5)P2. Upon opening of the channel, lysosomal Ca2+ would diffuse and trigger phagosome-lysosome fusion since Ca2+ is known to induce membrane fusion post-docking of SNARE proteins. In addition, we also demonstrated that the lipid kinase PIKfyve coordinates the neutrophils immune response by controlling phagosome maturation and regulating Rac GTPase activity. PIKfyve produces both PI(3,5)P2 and phosphatidylinositol-5-phosphate (PI5P); therefore, it might control phagosome maturation through production of PI(3,5)P2 and activation of TRPML1 as well as regulates ROS production and chemotaxis through synthesis of PI5P, which leads to the activation of Tiam1, and Rac GTPase.

Macrophage morphological plasticity and migration is Rac signalling and MMP9 dependant

In vitro, depending on extracellular matrix (ECM) architecture, macrophages migrate either in amoeboid or mesenchymal mode; while the first is a general trait of leukocytes, the latter is associated with tissue remodelling via Matrix Metalloproteinases (MMPs). To assess whether these stereotyped migrations could be also observed in a physiological context, we used the zebrafish embryo and monitored macrophage morphology, behaviour and capacity to mobilise haematopoietic stem/progenitor cells (HSPCs), as a final functional readout. Morphometric analysis identified 4 different cell shapes. Live imaging revealed that macrophages successively adopt all four shapes as they migrate through ECM. Treatment with inhibitors of MMPs or Rac GTPase to abolish mesenchymal migration, suppresses both ECM degradation and HSPC mobilisation while differently affecting macrophage behaviour. This study depicts real time macrophage behaviour in a physiological context and reveals extreme reactivity of these cells constantly adapting and switching migratory shapes to achieve HSPCs proper mobilisation.

Macrophage Plasticity is Rac Signalling and MMP9 Dependant

In vitro, depending on extracellular matrix (ECM) architecture, macrophages migrate either in amoeboid or mesenchymal mode; while the first is a general trait of leukocytes, the latter is associated with tissue remodelling via Matrix Metalloproteinases (MMPs). To assess whether these stereotyped migrations could be also observed in a physiological context, we used the zebrafish embryo and monitored macrophage morphology, behaviour and capacity to mobilisation haematopoietic stem/progenitor cells (HSPCs), as a final functional readout. Morphometric analysis identified 4 different cell shapes. Live imaging revealed that macrophages successively adopt all four shapes as they migrate through ECM. Treatment with inhibitors of MMPs or Rac GTPase to abolish mesenchymal migration, suppresses both ECM degradation and HSPC mobilisation while differently affecting macrophage behaviour. This study depicts real time macrophage behaviour in a physiological context and reveals extreme reactivity of these cells constantly adapting and switching migratory shapes to achieve HSPCs proper mobilisation.

A Progressive Loss of phosphoSer138-Profilin Aligns with Symptomatic Course in the R6/2 Mouse Model of Huntington’s Disease: Possible Sex-Dependent Signaling

The R6/2 transgenic mouse model of Huntington’s disease (HD) carries several copies of exon1 of the huntingtin gene that contains a highly pathogenic 120 CAG-repeat expansion. We used kinome analysis to screen for kinase activity patterns in neural tissues from wildtype (WT) and R6/2 mice at a pre-symptomatic (e.g., embryonic) and symptomatic (e.g., between 3 and 10 weeks postnatal) time points. We identified changes in several signaling cascades, for example, the Akt/FoxO3/CDK2, mTOR/ULK1, and RAF/MEK/CREB pathways. We also identified the Rho-Rac GTPase cascade that contributes to cytoskeleton organization through modulation of the actin-binding proteins, cofilin and profilin. Immunoblotting revealed higher levels of phosphoSer138-profilin in embryonic R6/2 mouse samples (cf. WT mice) that diminish progressively and significantly over the postnatal, symptomatic course of the disease. We detected sex- and genotype-dependent patterns in the phosphorylation of actin-regulators such a ROCK2, PAK, LIMK1, cofilin, and SSH1L, yet none of these aligned consistently with the changing levels of phosphoSer138-profilin. This could be reflecting an imbalance in the sequential influences these regulators are known to exert on actin signaling. The translational potential of these observations was inferred from preliminary observations of changes in LIMK-cofilin signaling and loss of neurite integrity in neural stem cells derived from an HD patient (versus a healthy control). Our observations suggest that a pre-symptomatic, neurodevelopmental onset of change in the phosphorylation of Ser138-profilin, potentially downstream of distinct signaling changes in male and female mice, could be contributing to cytoskeletal phenotypes in the R6/2 mouse model of HD pathology.