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Rac GTPase activating protein 1 (RACGAP1) has been characterized in the pathogenesis and progression of several malignancies, however, little is known regarding its role in the development of gallbladder cancer (GBC). This investigation seeks to describe the role of RACGAP1 and its associated molecular mechanisms in GBC. It was found that RACGAP1 was highly expressed in human GBC tissues, which was associated to poorer overall survival (OS). Gene knockdown of RACGAP1 hindered tumor cell proliferation and survival both in vitro and in vivo. We further identified that RACGAP1 was involved in DNA repair through its binding with DNA ligase 3 (LIG3), a crucial component of the alternative-non-homologous end joining (Alt-NHEJ) pathway. RACGAP1 regulated LIG3 expression independent of RhoA activity. RACGAP1 knockdown resulted in LIG3-dependent repair dysfunction, accumulated DNA damage and Poly(ADP-ribosyl) modification (PARylation) enhancement, leading to increased apoptosis and suppressed cell growth. We conclude that RACGAP1 exerts a tumor-promoting role via binding LIG3 to reduce apoptosis and facilitate cell growth in GBC, pointing to RACGAP1 as a potential therapeutic target for GBC.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease, so there is an urgent need to suppress its development at early stage. Shenkang pills (SKP) are a hospital prescription selected and optimized from effective traditional Chinese medicinal formulas for clinical treatment of DN. In the present study, liquid chromatography-quadrupole-time of flight-mass spectrometry (LC-Q-TOF-MS) and total contents qualification were applied to generate a quality control standard of SKP. For verifying the therapeutic effects of SKP, db/db mice were administered intragastrically with SKP at a human-equivalent dose (1.82 g/kg) for 4 weeks. Moreover, the underlying mechanism of SKP were analyzed by the renal RNA sequencing and network pharmacology. LC-Q-TOF-MS identified 46 compounds in SKP. The total polysaccharide and organic acid content in SKP were 4.60 and 0.11 mg/ml, respectively, while the total flavonoid, saponin, and protein content were 0.25, 0.31, and 0.42 mg/ml, respectively. Treatment of SKP significantly reduced fasting blood glucose, improved renal function, and ameliorated glomerulosclerosis and focal foot processes effacement in db/db mice. In addition, SKP protected podocytes from injury by increasing nephrin and podocin expression. Furthermore, transcriptome analyses revealed that 430 and 288 genes were up and down-regulated in mice treated with SKP, relative to untreated controls. Gene ontology enrichment analysis revealed that the differentially expressed genes mainly involved in modulation of cell division and chromosome segregation. Weighted gene co-expression network analysis and network pharmacology analysis indicated that aurora kinase B (AURKB), Rac GTPase activating protein 1 (RacGAP1) and SHC binding, and spindle associated 1 (shcbp1) might be the core targets of SKP. This protein and Ras homolog family member A (RhoA) were found overexpression in db/db mice, but significantly decreased with SKP treatment. We conclude that SKP can effectively treat early-stage DN and improve renal podocyte dysfunction. The mechanism may involve down-regulation of the AURKB/RacGAP1/RhoA pathway.

WOS: 000382974300005 PubMed ID: 27284123 Objectives. The aims of this study are to evaluate expressions of Ki67, RacGAP1 (MgcRacGAP) and topoisomerase 2 alpha (TOP2a), the markers related with cell proliferation that have been proposed to affect the prognosis in the literature and correlate the results with clinicopathological parameters of breast cancer patients. Methods. Ki67, RacGAP1, and TOP2a antibodies were applied immunohistochemically to the tissue micrarray blocks of 457 female breast cancer patients. The results were correlated with clinical, prognostic, histopathological features, and other immunohistochemical findings (estrogen receptor [ER], progesterone receptor [PR], HER2, cytokeratin [CK]5/6, CK14, epidermal growth factor receptor [EGFR] and vimentin), statistically. Results. Ki67 expression demonstrated direct correlation with TOP2a expression, mitotic count, tumor grade, geographic necrosis, basal-like phenotype. RacGAP1 expression was directly correlated with TOP2a expression, nipple invasion, and number of metastatic lymph nodes, and it was inversely correlated with PR expression. TOP2a expression was directly correlated with vimentin and Ki67 expressions, mitotic count, tumor grade, and geographic necrosis, and nipple invasion, and negatively correlated with ER and PR expressions. Higher TOP2a and Ki67 expressions were correlated with shorter overall survival. Higher TOP2a expression and RacGAP1 positivity were directly correlated with shorter disease-free survival. Conclusion. This study showed that the overexpressions of Ki67, RacGAP1, and TOP2a affect the prognosis adversely, thus to develop target therapies against RacGAP1 and TOP2a as well as using Ki67 as a part of routine pathology practice might be beneficial in breast cancer therapy and prediction of prognosis. Bozok University Scientific Research Projects Unit [2014TF/A92] The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported financially by Bozok University Scientific Research Projects Unit (Project No. 2014TF/A92).

Accumulating studies have implicated the role of long non-coding RNAs (lncRNAs) in the pathogenesis of hepatocellular carcinoma (HCC) through the regulating transcription and mRNA stability. A recent report has linked Rac GTPase-activating protein 1 (RACGAP1) to the early recurrence of HCC. The current study aimed to ascertain whether MAGI2 antisense RNA 3 (MAGI2-AS3) influences the development of HCC by regulating RACGAP1. MAGI2-AS3 expression was initially quantified in both the HCC tissues and cell lines. In order to elucidate the role of MAGI2-AS3 in the development of HCC, MAGI2-AS3 was overexpressed or silenced in HCC cells after which cell proliferation, apoptosis, invasion, and migration were evaluated. Chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), and biotin-labeled RNA pull-down assays were conducted to determine the interactions among MAGI2-AS3, KDM1A, and RACGAP1. Finally, the effects of MAGI2-AS3 and RACGAP1 on the tumorigenesis of transplanted HCC cells in nude mice were evaluated. MAGI2-AS3 was found to be under-expressed in HCC tissues and cell lines. The restoration of MAGI2-AS3 was identified to markedly inhibit HCC cell growth, migrating ability, and invasiveness, and promote cell apoptosis. Interaction between MAGI2-AS3 and KDM1A was identified. KDM1A recruited by MAGI2-AS3 was found to promote H3K4me2 demethylation at the RACGAP1 promoter, which ultimately decreased the expression of RACGAP1. We also identified that RACGAP1 knockdown eliminated the stimulatory effects of MAGI2-AS3 silencing on the malignant phenotypes of HCC cells. Additionally, the expression of MAGI2-AS3 reduced tumor weight and size in HCC transplanted nude mice. Taken together, the key observations of the current study demonstrate the potential of MAGI2-AS3 as a tumor suppressor and a promising target for HCC treatment. Keywords: hepatocellular carcinoma, MAGI2-AS3, KDM1A, RACGAP1, demethylation, H3K4me2

RCP-driven α5β1 recycling suppresses Rac activity through the RacGAP1–IQGAP1 complex to permit local activation of RhoA and drive invasive migration., Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)–dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.

Purpose: RACGAP1 is a Rac GTPase-activating protein involved in cell growth regulation, cell transformation and metastasis. The aim of the present study was to explore the prognostic and/or predictive significance of RACGAP1 mRNA expression on disease-free survival (DFS) and overall survival (OS) in high-risk early breast cancer patients and compare it to that of Ki67 protein expression and to the Nottingham prognostic index (NPI). Methods: A total of 595 high-risk breast cancer patients were treated in a two-arm trial evaluating postoperative dose-dense sequential chemotherapy with epirubicin followed by CMF with or without paclitaxel. RNA was extracted from 314 formalin-fixed paraffin-embedded primary tumor tissue samples followed by one-step quantitative RT-PCR for assessing RACGAP1 mRNA expression. Results: High RACGAP1 mRNA expression (above the median) was associated with poor DFS (log-rank, p = 0.002) and OS (p < 0.001). High histological grade, as well as high Ki67 protein expression, was more frequent in the high-expression group of RACGAP1. Results of the Cox multivariate regression analysis revealed that high RACGAP1 mRNA expression independently predicted poor overall survival (Wald's p = 0.008). High Ki67 protein expression was also an adverse prognostic factor for death (p = 0.016), while high NPI score values were not. Conclusions: High RACGAP1 mRNA expression, as assessed by qRT-PCR, was found to be of adverse prognostic significance in high-risk early breast cancer patients treated with dose-dense sequential chemotherapy. The utility of RACGAP1 mRNA expression in patient selection for treatment with aggressive chemotherapy regimens should be further explored and validated in larger cohorts. © 2012 Springer-Verlag Berlin Heidelberg. 71 1 245 255

Previously, we established a microRNA (miRNA) expression signature in pancreatic ductal adenocarcinoma (PDAC) tissues using RNA sequencing and found significantly reduced expression of miR-204-5p. Here, we aimed to investigate the functional significance of miR-204-5p and to identify miR-204-5p target genes involved in PDAC pathogenesis. Cancer cell migration and invasion were significantly inhibited by ectopic expression of miR-204-5p in PDAC cells. Comprehensive gene expression analyses and in silico database searches revealed 25 putative targets regulated by miR-204-5p in PDAC cells. Among these target genes, high expression levels of RACGAP1, DHRS9, AP1S3, FOXC1, PRP11, RHBDL2 and MUC4 were significant predictors of a poor prognosis of patients with PDAC. In this study, we focused on RACGAP1 (Rac guanosine triphosphatase-activating protein 1) because its expression was most significantly predictive of PDAC pathogenesis (overall survival rate: p = 0.0000548, disease-free survival rate: p = 0.0014). Overexpression of RACGAP1 was detected in PDAC clinical specimens, and its expression enhanced the migration and invasion of PDAC cells. Moreover, downstream genes affected by RACGAP1 (e.g., MMP28, CEP55, CDK1, ANLN and S100A14) are involved in PDAC pathogenesis. Our strategy to identify antitumor miRNAs and their target genes will help elucidate the molecular pathogenesis of PDAC.

// Elisa Neubauer 1 , Ralph M. Wirtz 2 , Daniel Kaemmerer 3 , Maria Athelogou 4 , Lydia Schmidt 1 , Jorg Sanger 5 , Amelie Lupp 1 1 Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University, Jena, Germany 2 STRATIFYER Molecular Pathology GmbH Koln, Koln, Germany 3 Department of General and Visceral Surgery, Zentralklinik Bad Berka, Bad Berka, Germany 4 DEFINIENS AG, Munchen, Germany 5 Laboratory of Cytology and Pathology, Bad Berka, Germany Correspondence to: Elisa Neubauer, email: elisa.specht@med.uni-jena.de Keywords: Ki-67, topoisomerase 2 alpha, RacGAP1, lung neuroendocrine neoplasms, IHC/RT-qPCR Received: March 13, 2016 Accepted: May 16, 2016 Published: May 31, 2016 ABSTRACT The classification of bronchopulmonary neuroendocrine neoplasms (BP-NEN) into four tumor entities (typical carcinoids (TC), atypical carcinoids (AC), small cell lung cancers (SCLC), large cell neuroendocrine lung carcinomas (LCNEC)) is difficult to perform accurately, but important for prognostic statements and therapeutic management decisions. In this regard, we compared the expression of three proliferation markers, Ki-67, Topoisomerase II alpha (TOP2A), and RacGAP1, in a series of tumor samples from 104 BP-NEN patients (24 TC, 21 AC, 52 SCLC, 7 LCNEC) using different evaluation methods (immunohistochemistry (IHC): Average evaluation, Hotspot evaluation, digital image analysis; RT-qPCR). The results indicated that all three markers had increased protein and mRNA expression with poorer differentiation and correlated well with each other, as well as with grading, staging, and poor survival. Compared with Ki-67 and TOP2A, RacGAP1 allowed for a clearer prognostic statement. The cut-off limits obtained for Ki-67-Average (IHC) were TC-AC 1.5, AC-SCLC 19, and AC-LCNEC 23.5. The Hotspot evaluation generated equal to higher, the digital image analysis generally lower between-entity cut-off limits. All three markers enabled a clear-cut differentiation between the BP-NEN entities, and all methods evaluated were suitable for marker assessment. However, to define optimal cut-off limits, the Ki-67 evaluation methods should be standardized. RacGAP1 appeared to be a new marker with great potential.

Canonical Wnt signaling is involved in gastric carcinogenesis. The aim of this study was to identify the link between Wnt signaling and aurora kinase A (AURKA), a target for the treatment of gastrointestinal cancers. Publicly available microarray data were used to identify phenotype‐specific protein–protein interaction (PPI) subnetworks. The in silico analysis revealed a gastric cancer‐specific PPI subnetwork consisting of 2745 proteins and 50,935 interactions. We focused on the link of AURKA to a Wnt‐specific interaction module consisting of 92 proteins. There was a direct association of AURKA with Rac GTPase‐activating protein 1 (RACGAP1), as well as with CTNBB1 (β‐catenin) and CDKN1A as second‐order interactors. Differential expression analysis revealed a significant downregulation of both AURKA and RACGAP1 in gastric cancer compared to noncancer controls. Biopsies from a prospective cohort of 56 patients with gastric cancer (32 intestinal type, 24 diffuse type) and 20 noncancer controls were used for validation of the identified targets. The RT‐PCR data confirmed a strong correlation of AURKA and RACGAP1 gene expression both in the tumor, the tumor‐adjacent and the tumor‐distant mucosa. RACGAP1 in the tumor was also associated with CTNBB1 expression, and inversely associated with CDKN1A gene expression. Immunohistochemistry confirmed expression of the RACGAP1 protein in gastric cancer and the tumor‐adjacent mucosa. RACGAP1 expression was not associated with tumor stage, grading, Lauren type, Helicobacter pylori infection, or age. In conclusion, AURKA is directly associated with the expression of RACGAP1, a modulator of the canonical Wnt signaling pathway.