Journal: Cancer research

Article Title: HACE1 prevents lung carcinogenesis via inhibition of RAC-family GTPases

doi: 10.1158/0008-5472.CAN-19-2270

Figure Lengend Snippet: (A) Amino acid (aa) sequence alignments of murine RAC1, RAC2, and RAC3. Amino acids highlighted in red indicate differences among the family members. The amino acid positions are indicted. (B) Relative mRNA expression of Rac1, Rac2 and Rac3 normalized to β-Actin expression in primary lung tumor cells, isolated from KRasG12D;Hace1+/+Rac1+/+Rac2+/+ mice (n=5) at week 7 post lung cancer induction, followed by RT-qPCR analysis. (C) In vitro ubiquitylation assay. Recombinant GST-HACE1 was incubated with GTP- or GDP-preloaded His-tagged RAC1 or RAC2 in the presence of E1, E2 (Ubch7), ubiquitin and ATP. As a control, catalytic dead HACE1C876S was used. Blots show RAC1 and RAC2 (detected via the His-tag), HACE1 and ubiquitin after 3 h incubation. Ubiquitylated RAC1 and RAC2 are indicated (white arrows). (D) Kaplan-Meier survival curves of KRasG12D;Hace1+/+Rac1+/+Rac2+/+ (n=23), KRasG12D;Hace1–/– (n=15), KRasG12D;Rac1fl/fl (n=25), KRasG12D;Hace1–/–Rac1fl/fl (n=19), KRasG12D;Rac2–/– (n=23) and KRasG12D;Hace1–/–Rac2–/– (n=7), KRasG12D;Rac1fl/flRac2–/– (n=19) and KRasG12D;Hace1–/–Rac1fl/flRac2–/– (n=20) mice. Mice were intratracheally instilled with Adeno-Cre virus on the indicated day 0. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 (log-rank test). (E) Representative H&E stained-lung sections and (F) tumor-to-lung ratios at week 18 post lung cancer induction for KRasG12D;Hace1+/+Rac1+/+Rac2+/+, KRasG12D;Rac1fl/fl, KRasG12D;Hace1–/–Rac1fl/fl, KRasG12D;Rac1fl/flRac2–/– and KRasG12D;Hace1–/–Rac1fl/flRac2–/– mice. Scale bars, 1 mm for 10x images and 50μm for 40x images of lung sections. * P<0.05, ** P<0.01 (One-way ANOVA, Tukey’s post-hoc test, n≥5 mice per cohort). Data in (B) and (F) are presented as mean values ± SEM.

Article Snippet: In vitro ubiquitylation assay His-tagged recombinant human RAC1 (Cytoskeleton, RC01) and RAC2 (Cytoskeleton, RC02) were preloaded with GTP or GDP as described ( 41 ).

Techniques: Sequencing, Expressing, Isolation, Quantitative RT-PCR, In Vitro, Ubiquitin Assay, Recombinant, Incubation, Staining

Journal: Cancer research

Article Title: HACE1 prevents lung carcinogenesis via inhibition of RAC-family GTPases

doi: 10.1158/0008-5472.CAN-19-2270

Figure Lengend Snippet: (A) Kaplan-Meier curves of overall survival and (B) disease-free survival for lung adenocarcinoma patients, based on HACE1 and RAC1 mRNA expression. (C) Kaplan-Meier curves of overall survival and (D) disease-free survival for lung adenocarcinoma patients, based on HACE1, RAC1 and RAC2 mRNA expression. (E) Schematic representation of genetic alterations in HACE1, RAC1, RAC2 and RAC3 in lung adenocarcinoma patients from the TCGA (PanCancer Atlas) data set for 507 cases. Color coding indicates mutation types: red, amplification; blue, homozygous deletion; yellow, missense mutation; black, truncating mutation. Percentages (%) of cases with alteration in HACE1, RAC1, RAC2 and RAC3 are indicated. Only altered cases are shown. (F) Heatmap of gene expression profiles of HACE1, RAC1, RAC2 and RAC3 in lung adenocarcinoma patients. Each row represents the expression of either HACE1, RAC1, RAC2 or RAC3. Each line corresponds to one lung cancer patient. Z-score (RNA Seq V2 RSEM) is shown from 10 (red, highest expression) to -2 (blue, lowest expression). The mRNA expression level in a single sample is depicted according to the color scale. (G) Correlation matrix showing Spearman’s rank correlation of HACE1, RAC1, RAC2 and RAC3 mRNA expression profiles. Correlation coefficients are shown in white and the associated p-values in black (statistically significant values with P<0.05 in bold). Orange and blue colors indicate positive and negative correlations, respectively, beige indicates no correlation.

Article Snippet: In vitro ubiquitylation assay His-tagged recombinant human RAC1 (Cytoskeleton, RC01) and RAC2 (Cytoskeleton, RC02) were preloaded with GTP or GDP as described ( 41 ).

Techniques: Expressing, Mutagenesis, Amplification, RNA Sequencing Assay

Journal: Cancer research

Article Title: HACE1 prevents lung carcinogenesis via inhibition of RAC-family GTPases

doi: 10.1158/0008-5472.CAN-19-2270

Figure Lengend Snippet: HACE1 ubiquitylates GTP-RAC1 when bound to the NADPH oxidase complex, leading to RAC1 degradation and thereby controlling ROS production (top, left). HACE1 deficiency results in an accumulation of GTP-bound RAC1, increased NADPH oxidase activity and enhanced levels of genotoxic cellular ROS, promoting cancer progression (top, right). Additionally, deregulated RAC1 could promote tumor development by ROS-independent mechanisms. In the absence of the more abundant RAC1, the activity of GTP-RAC2 when bound to the NADPH oxidase complex is controlled by HACE1, leading to decreased cellular ROS levels (bottom, left). When HACE1 and RAC1 are both ablated, active GTP-RAC2 can compensate and promote cancer progression (bottom, right).

Article Snippet: In vitro ubiquitylation assay His-tagged recombinant human RAC1 (Cytoskeleton, RC01) and RAC2 (Cytoskeleton, RC02) were preloaded with GTP or GDP as described ( 41 ).

Techniques: Activity Assay

Journal: Haematologica

Article Title: A gain-of-function RAC2 mutation is associated with bone marrow hypoplasia and an autosomal dominant form of severe combined immunodeficiency

doi: 10.3324/haematol.2019.230250

Figure Lengend Snippet: Identification and effects of the p.G12R RAC2 mutation on the GTPase activity of RAC2. (A) Pedigrees of the three patients from two unrelated kindred. Black boxes and black circles respectively represent the affected male (P1) and affected females (P2, P3). White boxes and circles respectively represent unaffected males and females. Arrows represent the probands and a double horizontal bar represents consanguinity. (B) A representative electropherogram of RAC2 DNA sequencing for control cells and patient cells, showing the c.34G>A mutation. (C) A representative immunoblot of RAC2 protein expression in lysates from control fibroblasts (Ctrl) and fibroblasts derived from the affected individuals (P1, P2, and P3). The loading control corresponds to GAPDH expression. (D) 3D models of the RAC2 G12R mutant. The structures are shown with GDP (left panel) or GTP (right panel) in the G1 binding pocket. For each model, a close-up view of the GDP/GTP binding pocket (dotted brown circle) is shown below the overall view. The figure was generated with the pymol program ( www.pymol.org/ ). (E) HEK293T cells were either not transduced (NT, control) or transduced with a lentiviral empty vector (WPI) containing the wild-type (WT) form of RAC2 cDNA, the mutated form described here (G12R) or (as a positive control) the constitutively activated GTP-bound RAC2 form (G12V). Two days after transduction, cells were recovered for analysis using the G-LISA assay (15 μg of total protein per well) for the quantification of the GTP-bound RAC2 form (RAC2 GTP). The results come from three independent experiments, and the table below the graph represents the mean of the percentage of GFP expressing cells (GFP + ) in the three independent experiments. *** P <0.001; **** P <0.0001.

Article Snippet: Levels of activated RAC2 were determined using the G-LISA ® RAC Activation Assay Biochem KitTM (#BK125, Cytoskeleton Inc.) according to the manufacturer’s instructions, except that RAC2 monoclonal specific antibody (AT2G11, sc-517424 Santa Cruz Biotechnology, Inc.) and HRPconjugated anti-mouse antibody (#1721011, Bio-Rad) were used to detect the amount of captured active RAC2 (details in the Online Supplementary Methods ).

Techniques: Mutagenesis, Activity Assay, DNA Sequencing, Control, Western Blot, Expressing, Derivative Assay, Binding Assay, Generated, Transduction, Plasmid Preparation, Positive Control

Journal: Applied Immunohistochemistry & Molecular Morphology

Article Title: Differentially Expressed Genes in Matched Normal, Cancer, and Lymph Node Metastases Predict Clinical Outcomes in Patients With Breast Cancer

doi: 10.1097/PAI.0000000000000717

Figure Lengend Snippet: In accordance with RNA-Seq data, mRNA expression of RAC2 and PTGDS by quantitative real-time PCR was upregulated in LNs compared with primary cancers. LN indicates lymph node metastasis; N, normal breast; PCR, polymerase chain reaction; T, cancer.

Article Snippet: The following probes of TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: Hs00427439_g1 ( RAC2 ), Hs00168748_m1 ( PTGDS ), and Hs02758991_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH ).

Techniques: RNA Sequencing, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Applied Immunohistochemistry & Molecular Morphology

Article Title: Differentially Expressed Genes in Matched Normal, Cancer, and Lymph Node Metastases Predict Clinical Outcomes in Patients With Breast Cancer

doi: 10.1097/PAI.0000000000000717

Figure Lengend Snippet: Representative immunostaining patterns of RAC2 and PTGDS in normal epithelium of the breast (A, D), corresponding carcinoma (B, E), and metastatic lymph node (C, F) (A–C, RAC2; D–F, PTGDS) (×400, scale bars; 60 μm).

Article Snippet: The following probes of TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: Hs00427439_g1 ( RAC2 ), Hs00168748_m1 ( PTGDS ), and Hs02758991_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH ).

Techniques: Immunostaining

Journal: Applied Immunohistochemistry & Molecular Morphology

Article Title: Differentially Expressed Genes in Matched Normal, Cancer, and Lymph Node Metastases Predict Clinical Outcomes in Patients With Breast Cancer

doi: 10.1097/PAI.0000000000000717

Figure Lengend Snippet: Distribution of Allred scores for RAC2 and PTGDS in normal breast tissues, corresponding carcinoma, and LN. The ends of the box represent the 25th and 75th percentiles; the bars indicate the 10th and 90th percentiles, and a horizontal line inside the box shows the median. LN indicates lymph node metastasis; N, normal breast; T, cancer.

Article Snippet: The following probes of TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: Hs00427439_g1 ( RAC2 ), Hs00168748_m1 ( PTGDS ), and Hs02758991_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH ).

Techniques:

Journal: Applied Immunohistochemistry & Molecular Morphology

Article Title: Differentially Expressed Genes in Matched Normal, Cancer, and Lymph Node Metastases Predict Clinical Outcomes in Patients With Breast Cancer

doi: 10.1097/PAI.0000000000000717

Figure Lengend Snippet: Overall survival analysis based on RAC2 expression.

Article Snippet: The following probes of TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: Hs00427439_g1 ( RAC2 ), Hs00168748_m1 ( PTGDS ), and Hs02758991_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH ).

Techniques: Expressing

Journal: eLife

Article Title: A novel Drosophila injury model reveals severed axons are cleared through a Draper/MMP-1 signaling cascade

doi: 10.7554/eLife.23611

Figure Lengend Snippet: Annokey analysis results of upregulated genes associated with glial membrane expansion and movement. (a) List of terms used in Annokey analysis. (b) Top ten hits associated with our Annokey key terms in Drosophila , mouse, and human. MMP-1/MMP-14 (red bold) is included in the top ten list for each species. DOI: http://dx.doi.org/10.7554/eLife.23611.011 10.7554/eLife.23611.012 Table 1—source data 1. Human HTML Annokey search results that include hyperlinks to NCBI Gene, GeneRIF and Pubmed databases. DOI: http://dx.doi.org/10.7554/eLife.23611.012 10.7554/eLife.23611.013 Table 1—source code 1. Drosophila HTML Annokey results, including hyperlinks to NCBI gene, GeneRIF, and PubMed databases. DOI: http://dx.doi.org/10.7554/eLife.23611.013 10.7554/eLife.23611.014 Table 1—source code 2. Mouse HTML Annokey results, including hyperlinks to NCBI Gene, GeneRIF,pu and PubMed databases. DOI: http://dx.doi.org/10.7554/eLife.23611.014 10.7554/eLife.23611.015 Table 1—source code 3. Human HTML Annokey results, including hyperlinks to NCBI Gene, GeneRIF, and PubMed databases. DOI: http://dx.doi.org/10.7554/eLife.23611.015

Article Snippet: Additional TaqMan gene expression assays were utilized: Ets21c-Dm01814139_m1; PGRP-SA-Dm01837990_g1; MMP-1-Dm01820359_m1; Relish-Dm02134843_g1; Ninjurin A-Dm01798347_g1; Hairy- Dm01822363_m1; Rac2-Dm01840631_s1; Cactus-Dm01807760_m1; Notch-Dm01841974_g1; CG6277 – Dm02369365_s1.

Techniques: Membrane