Expression Of Rab7 Research Articles

Rab23 Regulates Radial Migration of Projection Neurons via N-cadherin

Radial migration of cortical projection neurons is a prerequisite for shaping a distinct multilayered cerebral cortex during mammalian corticogenesis. Members of Rab GTPases family were reported to regulate radial migration. Here, in vivo conditional knockout or in utero knockdown (KD) of Rab23 in mice neocortex causes aberrant polarity and halted migration of cortical projection neurons. Further investigation of the underlying mechanism reveals down-regulation of N-cadherin in the Rab23-deficient neurons, which is a cell adhesion protein previously known to modulate radial migration. (Shikanai M, Nakajima K, Kawauchi T. 2011. N-cadherin regulates radial glial fiber-dependent migration of cortical locomoting neurons. Commun Integr Biol. 4:326–330.) Interestingly, pharmacological inhibition of extracellular signal-regulated kinases (ERK1/2) also decreases the expression of N-cadherin, implicating an upstream effect of ERK1/2 on N-cadherin and also suggesting a link between Rab23 and ERK1/2. Further biochemical studies show that silencing of Rab23 impedes activation of ERK1/2 via perturbed platelet-derived growth factor-alpha (PDGFRα) signaling. Restoration of the expression of Rab23 or N-cadherin in Rab23-KD neurons could reverse neuron migration defects, indicating that Rab23 modulates migration through N-cadherin. These studies suggest that cortical neuron migration is mediated by a molecular hierarchy downstream of Rab23 via N-cadherin.

Rab23 promotes the cisplatin resistance of ovarian cancer via the Shh-Gli-ABCG2 signaling pathway.

As a novel member of the Rab GTPase family, the role of Rab23 has been reported in multiple types of tumor. However, to the best of our knowledge, the role of Rab23 in ovarian cancer (OC) has not yet been reported. In the present study, immunohistochemistry analysis demonstrated that Rab23 was upregulated in OC tissue; survival analysis indicated that Rab23 expression was associated with a reduced overall survival (OS) rate and disease-free survival (DFS) time. In vitro experiments also demonstrated the increased expression of Rab23 in the OC cells lines, A2780 and SKOV-3, compared with in the normal ovarian cell line, IOSE80. Following the silencing of ABCG2 in SKOV-3 cells, ATP-binding cassette sub-family G member 2 (ABCG2) expression was significantly downregulated both at the RNA and protein levels. The cisplatin (DDP) IC50 declined from 43.09±7.12 µmol/l in control cells to 26.46±5.38 µmol/l in SKOV-3 cells with silenced Rab23. In contrast, in A2780 cells overexpressing Rab23 (A2780-Rab23), ABCG2 expression was significantly upregulated and the DDP IC50 increased from 27.42±6.54 µmol/l in control cells to 45.92±5.23 µmol/l in A2780-Rab23. Investigation into the potential molecular mechanisms for this revealed that the expression of sonic hedgehog (Shh) and Gli family zinc finger 1 (Gli1) was increased in A2780-Rab23 cells, whereas silencing Rab23 in SKOV-3 cells significantly inhibited the expression of Shh and Gli1. The Gli1 inhibitor GANT-61 significantly abrogated the increased ABCG2 expression in A2780-Rab23 cells. Furthermore, the DDP IC50 in A2780-Rab23 cells decreased significantly following the silencing of ABCG2 expression; the IC50 declined from 51.66±8.32 µmol/l in A2780-Rab23 cells to 25.61±6.17 µmol/l in A2780-Rab23 cells with silenced ABCG2. Collectively, the results indicate that Rab23 promotes the DDP resistance of OC cells via the Shh-Gli1-ABCG2 pathway, providing the proof of principle for the further investigation of drug resistance therapy targeting Rab23.

The level and clinical significance of Rab11 in children with sepsis

Objective To investigate the expression of Rab11 in children with sepsis at different stages and severe sepsis and its relationship with the occurrence and development of sepsis in children. Methods A prospective control study was performed.All cases were enrolled from Bao′an Maternal and Children Health Care Hospital, and they were divided into sepsis group(40 cases) who were diagnosed as sepsis, severe sepsis group(20 cases) with diagnosis of severe sepsis, and healthy control group(40 healthy children). Venous blood samples were collected at admission, and the expression level of blood leukocyte Rab11 was determined by Western blot.In the sepsis group, the expression levels of Rab11 were evaluated at the initial, the extreme and the recovery stages of sepsis, and were compared with those in severe sepsis group and healthy control group, respectively.Spearman correlation analysis was used to evaluate the relationship between the expressions of Rab11 and the levels of some parameters in blood, including white blood cell, neutrophils, lymphocytes, monocytes, eosinophilic granulocyte, C-reactive protein and procalcitonin in blood, at the extreme stage.Meanwhile, the levels of Rab11 at extreme stage of sepsis, caused by different diseases, such as severe pneumonia, bronchiectasis complicated pulmonary infection, biliary tract infection, urinary tract infection, necrotizing enterocolitis and severe enteric viruses infection, were compared with each other. Results At the initial and the extreme stages of sepsis, as well as in severe sepsis group, the levels of Rab11 were significantly lower than that in the healthy control group(0.54 times, 0.23 times and 0.07 times, P 0.05). There was no relationship between the level of Rab11 and the number of white blood cell, neutrophils, lymphocytes, monocytes, eosinophilic granulocyte, while the level of Rab11 was negatively correlated with C-reactive protein(r=-0.58, P=0.014) and procalcitonin(r=-0.63, P=0.003) at the extreme stage of sepsis.There was no significant difference in the expression level of Rab11, at the extreme stage of sepsis, among those patients with severe pneumonia, bronchiectasis and pulmonary infection, biliary tract infection, urinary tract infection, necrotizing enterocolitis and severe enterovirus infection(P>0.05). Conclusion The level of Rab11 is differently expressed at different stages of sepsis, and could be used as a predictor of the severity of sepsis in children. Key words: Sepsis; Rab11; Expression

Abstract B089: MicroRNA (miR) 214-3p targets ras-related protein14 (RAB14) to inhibit cell migration and invasion in esophageal cancer cells

Abstract Objectives: MiR-214-3p acts as a tumor suppressor in various malignancies. We have previously shown that miR-214-3p is markedly downregulated in esophageal cancer cell lines compared to esophageal epithelial cells and its downregulation contributes to chemoresistance in esophageal cancer cells. In several miR-target sequence analysis programs, miR-214-3p is predicted to bind RAB14 mRNA with high affinity. RAB14 is a member of the ras-associated binding protein family of low molecular mass GTPases that are involved in membrane trafficking. Its role in cancer is not well known. The objective of this study was to determine expression of RAB14 in esophageal cancer cells as well as to investigate the interaction between miR-214-3p and RAB14 in these cells and to characterize the functional implications of this interaction. Methods: Studies were performed in human esophageal epithelial (hESO) cells and in FLO1, SKGT4, and TE7 human esophageal cancer cells. Expression of miR-214-3p and RAB14 mRNA in these cell lines was measured by real-time PCR. RAB14 protein expression levels were examined by Western blot. Function of miR-214-3p was tested through its overexpression and silencing. Association of miR-214-3p with RAB14 mRNA was established using a biotinylated RNA pull-down assay and was confirmed by luciferase reporter assay. Cellular proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. xCELLigence RTCA system was used to access cell migration and invasion in real time. Results: Levels of miR-214-3p in FLO1, SKGT4, and TE7 esophageal cancer cells are markedly reduced compared to hESO cells. RAB14 mRNA and protein expression are correspondingly elevated in these cells compared to hESO cells. RAB14 mRNA and protein expression levels decreased in a time-dependent manner following miR-214-3p overexpression in FLO1, SKGT4, and TE7 cells. In reciprocal experiments, silencing miR-214-3p in hESO cells resulted in increased RAB14 mRNA and protein levels. Direct interaction of miR-214-3p and RAB14 mRNA was confirmed by biotinylated RNA-pull down assay and luciferase reporter construct. The stability of RAB14 mRNA is decreased following ectopic expression of miR-214-3p in FLO1 cells. Forced expression of miR-214-3p in FLO1 and SKGT4 cells led to a marked decrease in cellular proliferation, migration, and invasion. Conclusions: MiR-214-3p expression is significantly reduced in FLO1, SKGT4, and TE7 esophageal cancer cells relative to hESO cells. MiR-214-3p directly binds and destabilizes RAB14 mRNA. Forced expression of miR-214-3p results in significantly decreased RAB14 mRNA and protein expression, leading to decreased cellular proliferation, migration, and invasion in esophageal cancer cells. These results add to the roles by which miR-214-3p functions as a tumor suppressor in esophageal cancer cells. Citation Format: Pornima Phatak, Jaladanki N. Rao, Carol B. Fowler, Douglas J. Turner, Jian-Ying Wang, James M. Donahue. MicroRNA (miR) 214-3p targets ras-related protein14 (RAB14) to inhibit cell migration and invasion in esophageal cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B089.

In vitro and in vivo effect of PD-1/PD-L1 blockade on microglia/macrophage activation and T cell subset balance in cryptococcal meningitis.

This study aimed to investigate the PD-1/ PD-L1 signaling pathway and its effects the activation of microglia/macrophage and balancing T cell subsets in cryptococcal meningitis (CM). A total of 126 CM patients and 126 healthy individuals were recruited for the study. The CM patients were treated with amphotericin B (AmB). Seventy five C57BL/6 mice were grouped into the normal control, CM model, CM + AmB, sham, and CM + PD-1 antibodies (Ab) groups. CD4+ and CD8+ T cells as well as microglia/macrophages were analyzed by means of flow cytometry. Ionized calcium-binding adaptor molecule 1 (Ibal) expression was detected using western blotting and immunohistochemistry techniques. And the expression of Rab5 and Rab11 were detected using an immunofluorescence assay. Both PD-1 and PD-L1 mRNA and protein expression among the mice in the study were evaluated by qRT-PCR and western blotting methods. Compared to the CM model group, the CM + AmB and CM + PD-1 Ab groups exhibited increased levels of Th1 cytokines and chemokines expression, and reduced levels of Th2 cytokines expressions. Elevated cell purity and viability of CD4+ T cell were recorded as well as increases in microglia, however, there were reductions in the number of CD8+ T cells. Depleted expressions of Ibal, Rab5, and Rab11 as well as reduced mRNA expressions of PD-1 and PD-L1 in CD4+ , microglia, and macrophage cells. The findings suggested that suppression of the PD-1/PD-L1 signaling pathway restricts the proliferation of CM by down-regulating the expressions of Th2 cells and suppressing microglia and macrophage activation.

Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer\u2019s disease resilience

BackgroundWhile age and the APOE ε4 allele are major risk factors for Alzheimer’s disease (AD), a small percentage of individuals with these risk factors exhibit AD resilience by living well beyond 75 years of age without any clinical symptoms of cognitive decline.MethodsWe used over 200 “AD resilient” individuals and an innovative, pedigree-based approach to identify genetic variants that segregate with AD resilience. First, we performed linkage analyses in pedigrees with resilient individuals and a statistical excess of AD deaths. Second, we used whole genome sequences to identify candidate SNPs in significant linkage regions. Third, we replicated SNPs from the linkage peaks that reduced risk for AD in an independent dataset and in a gene-based test. Finally, we experimentally characterized replicated SNPs.ResultsRs142787485 in RAB10 confers significant protection against AD (p value = 0.0184, odds ratio = 0.5853). Moreover, we replicated this association in an independent series of unrelated individuals (p value = 0.028, odds ratio = 0.69) and used a gene-based test to confirm a role for RAB10 variants in modifying AD risk (p value = 0.002). Experimentally, we demonstrated that knockdown of RAB10 resulted in a significant decrease in Aβ42 (p value = 0.0003) and in the Aβ42/Aβ40 ratio (p value = 0.0001) in neuroblastoma cells. We also found that RAB10 expression is significantly elevated in human AD brains (p value = 0.04).ConclusionsOur results suggest that RAB10 could be a promising therapeutic target for AD prevention. In addition, our gene discovery approach can be expanded and adapted to other phenotypes, thus serving as a model for future efforts to identify rare variants for AD and other complex human diseases.

Impaired placental autophagy in placental malaria.

BackgroundPlacental malaria is a major cause of low birthweight, principally due to impaired fetal growth. Intervillositis, a local inflammatory response to placental malaria, is central to the pathogenesis of poor fetal growth as it impairs transplacental amino acid transport. Given the link between inflammation and autophagy, we investigated whether placental malaria-associated intervillositis increased placental autophagy as a potential mechanism in impaired fetal growth.MethodsWe examined placental biopsies collected after delivery from uninfected women (n = 17) and from women with Plasmodium falciparum infection with (n = 14) and without (n = 7) intervillositis. Western blotting and immunofluorescence staining coupled with advanced image analysis were used to quantify the expression of autophagic markers (LC3-II, LC3-I, Rab7, ATG4B and p62) and the density of autophagosomes (LC3-positive puncta) and lysosomes (LAMP1-positive puncta).ResultsPlacental malaria with intervillositis was associated with higher LC3-II:LC3-I ratio, suggesting increased autophagosome formation. We found higher density of autophagosomes and lysosomes in the syncytiotrophoblast of malaria-infected placentas with intervillositis. However, there appear to be no biologically relevant increase in LC3B/LAMP1 colocalization and expression of Rab7, a molecule involved in autophagosome/lysosome fusion, was lower in placental malaria with intervillositis, indicating a block in the later stage of autophagy. ATG4B and p62 expression showed no significant difference across histological groups suggesting normal autophagosome maturation and loading of cargo proteins into autophagosomes. The density of autophagosomes and lysosomes in the syncytiotrophoblast was negatively correlated with placental amino acid uptake.ConclusionsPlacental malaria-associated intervillositis is associated with dysregulated autophagy that may impair transplacental amino acid transport, possibly contributing to poor fetal growth.

Endothelial Rab7 GTPase mediates tumor growth and metastasis in lysosomal acid lipase–deficient mice

Tumors depend on their microenvironment for sustained growth, invasion, and metastasis. In this environment, endothelial cells (ECs) are an important stromal cell type interacting with malignant cells to facilitate tumor angiogenesis and cancer cell extravasation. Of note, lysosomal acid lipase (LAL) deficiency facilitates melanoma growth and metastasis. ECs from LAL-deficient (lal-/-) mice possess enhanced proliferation, migration, and permeability of inflammatory cells by activating the mammalian target of rapamycin (mTOR) pathway. Here we report that lal-/- ECs facilitated in vivo tumor angiogenesis, growth, and metastasis, largely by stimulating tumor cell proliferation, migration, adhesion, and transendothelial migration via increased expression of IL-6 and monocyte chemoattractant protein 1 (MCP-1). This prompted us to look for lysosomal proteins that are involved in lal-/- EC dysfunctions. We found that lal-/- ECs displayed increased expression of Rab7, a late endosome/lysosome-associated small GTPase. Moreover, Rab7 and mTOR were co-increased and co-localized to lysosomes and physically interacted in lal-/- ECs. Rab7 inhibition reversed lal-/- EC dysfunctions, including decreasing their enhanced migration and permeability of tumor-stimulatory myeloid cells, and suppressed EC-mediated stimulation of in vitro tumor cell transmigration, proliferation, and migration and in vivo tumor growth and metastasis. Finally, Rab7 inhibition reduced overproduction of reactive oxygen species and increased IL-6 and MCP-1 secretion in lal-/- ECs. Our results indicate that metabolic reprogramming resulting from LAL deficiency enhances the ability of ECs to stimulate tumor cell proliferation and metastasis through stimulation of lysosome-anchored Rab7 activity.

Stapled peptide inhibitors of RAB25 target context-specific phenotypes in cancer

Recent evidence has established a role for the small GTPase RAB25, as well as related effector proteins, in enacting both pro-oncogenic and anti-oncogenic phenotypes in specific cellular contexts. Here we report the development of all-hydrocarbon stabilized peptides derived from the RAB-binding FIP-family of proteins to target RAB25. Relative to unmodified peptides, optimized stapled peptides exhibit increased structural stability, binding affinity, cell permeability, and inhibition of RAB25:FIP complex formation. Treatment of cancer cell lines in which RAB25 is pro-oncogenic with an optimized stapled peptide, RFP14, inhibits migration, and proliferation in a RAB25-dependent manner. In contrast, RFP14 treatment augments these phenotypes in breast cancer cells in which RAB25 is tumor suppressive. Transcriptional profiling identified significantly altered transcripts in response to RAB25 expression, and treatment with RFP14 opposes this expression profile. These data validate the first cell-active chemical probes targeting RAB-family proteins and support the role of RAB25 in regulating context-specific oncogenic phenotypes.

Role of Rab5 in the formation of macrophage-derived foam cell

BackgroundFoam cells play a key role in the occurrence and pathogenesis of atherosclerosis. Its formation starts with the ingestion of oxidized low-density lipoprotein (oxLDL). The process is associated with Ras related protein in brain 5 (Rab5) which plays a critical role in regulating endocytosis and early endosomal trafficking. Base on this, we presumed that Rab5 might participate in the maturation of foam cell. The aim of this study is to investigate the effect of Rab5 on macrophage cholesterol during the evolvement of macrophage when induced by oxLDL to the formation of foam cell.MethodsImmunohistochemistry was performed to analyze the distribution of macrophages and Rab5 in atherosclerotic plaque. RNA inteference study and transfection of inactive mutant (GFP-Rab5-S34N) and active mutant (GFP-Rab5-Q79L) in U937-derived macrophage were utilized to investigate the impact of Rab5 on the process of macrophage cholesterol, which could be detected by oil red O staining, determination of intracellular lipid content, filipin staining, nile red staining and the costaining of early endosome antigen-1 (EEA-1) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylin dicarbocyanine (Dil)-labelled oxLDL (Dil-oxLDL).ResultsRab5 was found abundantly localized in macrophage rich areas of human atherosclerotic lesions. On the foam cell study, the expression of Rab5 was increased after the incubation of oxLDL. The inteference study indicated the depletion of Rab5 led to the decreases of oil red O staining areas, total cholesterol and cholesterol esters in U937-derived marophages. Moreover, the fluorescence intensity of filipin and nile red staining were lower in GFP-Rab5-S34N as compared with GFP-Rab5-Q79L. The confocal study demonstrated less Dil-oxLDL was internalized in GFP-Rab5-S34N as compared with GFP-Rab5-Q79L; the result showed also the decrease in colocalization of internalized Dil-oxLDL and EEA-1 for GFP-Rab5-S34N as compared with GFP-Rab5-Q79L.ConclusionsRab5 plays an important role in modulating the intracellular cholesterol of macrophages and consequently mediating the formation of foam cells.

Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells.

During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses.IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that entry of JEV into BHK-21 cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow.

RABIF/MSS4 is a Rab-stabilizing holdase chaperone required for GLUT4 exocytosis

Rab GTPases are switched from their GDP-bound inactive conformation to a GTP-bound active state by guanine nucleotide exchange factors (GEFs). The first putative GEFs isolated for Rabs are RABIF (Rab-interacting factor)/MSS4 (mammalian suppressor of Sec4) and its yeast homolog DSS4 (dominant suppressor of Sec4). However, the biological function and molecular mechanism of these molecules remained unclear. In a genome-wide CRISPR genetic screen, we isolated RABIF as a positive regulator of exocytosis. Knockout of RABIF severely impaired insulin-stimulated GLUT4 exocytosis in adipocytes. Unexpectedly, we discovered that RABIF does not function as a GEF, as previously assumed. Instead, RABIF promotes the stability of Rab10, a key Rab in GLUT4 exocytosis. In the absence of RABIF, Rab10 can be efficiently synthesized but is rapidly degraded by the proteasome, leading to exocytosis defects. Strikingly, restoration of Rab10 expression rescues exocytosis defects, bypassing the requirement for RABIF. These findings reveal a crucial role of RABIF in vesicle transport and establish RABIF as a Rab-stabilizing holdase chaperone, a previously unrecognized mode of Rab regulation independent of its GDP-releasing activity. Besides Rab10, RABIF also regulates the stability of two other Rab GTPases, Rab8 and Rab13, suggesting that the requirement of holdase chaperones is likely a general feature of Rab GTPases.

Boosting efferocytosis in alveolar space using BCG vaccine to protect host against influenza pneumonia.

Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.

RIN1 promotes renal cell carcinoma malignancy by activating EGFR signaling through Rab25.

We previously identified the important role of RIN1 expression in the prognosis of clear cell renal cell carcinoma (ccRCC). The role of RIN1 in ccRCC malignancy and underlying molecular mechanisms remain unclear. Here we report that ccRCC cells and tissues expressed more RIN1 than normal controls. Gain‐of‐function and loss‐of‐function studies demonstrated that RIN1 enhanced ccRCC cell growth, migration and invasion abilities in vitro and promoted tumor growth and metastasis in vivo. Mechanistic studies revealed that RIN1 has an activating effect on EGFR signaling in ccRCC. In addition, we unveil Rab25, a critical GTPase in ccRCC malignancy, as a functional RIN1 interacting partner. Knockdown of Rab25 eliminated the augmentation of carcinoma cell proliferation, migration and invasion by ectopic RIN1. We also confirmed that RIN1 and Rab25 expression correlates with the overall‐survival of ccRCC patients from TCGA. These findings suggest that RIN1 plays an important oncogenic role in ccRCC malignancy by activation of EGFR signaling through interacting with Rab25, and RIN1 could be employed as an effective therapeutic target for ccRCC.

Abstract 1338: Exosome secretion promotes proliferation of African American prostate cancer cells under hypoxia: Role of HIF2A and RAB signaling

Abstract Background and Objectives: African American men experience worse prostate cancer (PCa) outcomes compared with those of Caucasian men not only in incidence and mortality rates but also harbor higher grade tumors compared to Caucasians. While the disparity is likely multifactorial, there may be biological basis for such phenomenon. Hypoxia in the tumor microenvironment is the key determinant of disease aggressiveness regulating primary growth, angiogenesis, metastasis and treatment outcomes. But role of hypoxia in the aggressiveness of PCa in African-American men has not been characterized yet. Here, we focused on hypoxia signaling, exosome biogenesis and characterization in African-American PCa cells compared to Caucasian PCa cells. Methods: We employed several techniques (cell viability assays, ultracentrifugation, nanoparticle tracking analyses [NTA], electron microscopy, and western blotting) to compare exosomes biogenesis and underlying molecular mechanisms in African-American and Caucasian PCa cells under normoxic and hypoxic conditions. Results: Compared to normoxic condition, African-American PCa cells (E006AA-hT and RC77T) either survived better or proliferated under hypoxia (1% O2), while growth of Caucasian PCa cells (PC3 and LNCaP) was strongly inhibited under hypoxia. NTA analyses showed that all PCa cells secreted more exosomes under hypoxia compared to normoxia, but more prominently in African-American PCa cells (E006AA-hT: 4.4 fold, RC77T: 35 fold, PC3: 3 fold and LNCaP: 2 fold). Exosomes secreted by E006AA-hT cells under hypoxia were loaded with higher amount of HSP70, HSP90, IL6 and Annexin II, and lower TGFβ. These exosomes also showed significantly higher MMP9 loading compared to E006AA-hT cell lysates. More importantly, under hypoxia, exosome biogenesis inhibition by GW4869 (20 µM) and 5-(N,N-dimethyl) amiloride (0.1-5 µg/ml) significantly reduced the E006AA-hT cell viability by 30% (p=0.0001) and 20-60% (p< 0.005 - 0.0001), respectively in 48 hrs. Further studies on hypoxia signaling revealed that E006AA-hT cells have significantly higher HIF2α and HIF1β but low HIF1α expression compared to LNCaP cells. Interestingly, HIF2α was present largely in nuclear fraction and its expression was similar under both normoxic and hypoxic conditions in E006AA-hT cells. Studies on the RAB and its effector proteins indicated that RabGAP5 expression was strongly increased under hypoxia in both E006AA-hT and LNCaP cells. Further, compared to LNCaP cells, E006AA-hT cells showed higher expression of Rab5 and Rabex5 but lower Rab4, and these molecules did not change significantly under hypoxia. Conclusion: African-American PCa cells have growth advantage under hypoxia mainly through higher exosome secretion; and higher expression/activity of HIF2α and RAB-signaling molecules could be associated with PCa aggressiveness in African-Americans. Citation Format: Gatikrushna Panigrahi, Taylor C. Peak, Sierra L. Patterson, Nicole Kasica, Ravi Singh, Ashok Hemal, Gagan Deep. Exosome secretion promotes proliferation of African American prostate cancer cells under hypoxia: Role of HIF2A and RAB signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1338. doi:10.1158/1538-7445.AM2017-1338

Abstract 5523: Loss of RAB25 cooperates with oncogenes in the transformation of human mammary epithelial cells

Abstract The RAB guanosine triphosphates (RAS-related in brain) belong to the Ras superfamily of GTPases, and loss of RAB 25 expression has been reported in a number of breast cancer cases containing H-Ras point mutations, particularly triple negative breast cancers (TNBC), one of the most aggressive subtypes of breast cancer and associated with a poor prognosis. The mechanism involved in the progression of these tumors is poorly understood. In this study, we are trying to understand if loss of RAB25 expression in Human Mammary Epithelial Cell (HMEC) lines co-operates with H-Ras mutations and contributes to tumorigenesis. HMEC were immortalized by transduction with LXSN CDK4 R24C, a mutant form of cyclin-dependent kinase, followed by transduction with hTERT, catalytic subunit of the telomerase enzyme that permits the cells to exceed the Hayflick Limit and become immortal. We have found that with loss of RAB25 and over expression of mutant H-Ras61L, immortal HMEC undergo transformation. We have looked into the co-operativity between loss of Rab25 and H-Ras61L mutant by in-vitro studies to show their anchorage independent growth and increased ability to migrate. Furthermore, cells express low CD24, high CD44, and very low levels of Claudin indicating that cells acquire stem-like properties upon transformation. Loss of RAB25 and over-expression of H-ras61L resulted in increased expression of transcription markers Snail and Slug that drive these cells to lose E-cadherin and undergo Epithelial Mesenchymal Transition (EMT). We also intend to carry out nude mice studies to look for the potential of these cells to form tumors and metastasize. This study shows that loss of RAB25 and over-expression of mutant H-Ras can transform HMEC and give rise to mesenchymal stem-like tumors. Our findings reveal that RAB25 functions as a tumor suppressor gene, and loss of RAB25 could serve as a novel biomarker in the prognosis of Claudin-low type of TNBC. Citation Format: Krishna Rao, Pooja Sridhar Joshi. Loss of RAB25 cooperates with oncogenes in the transformation of human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5523. doi:10.1158/1538-7445.AM2017-5523

High expression of Rab25 contributes to malignant phenotypes and biochemical recurrence in patients with prostate cancer after radical prostatectomy

BackgroundRas-related protein 25 (Rab25) functions either as an oncogene or a tumor suppressor with a cancer type-dependent manner. We aimed to investigate clinical significance of Rab25 in prostate cancer (PCa).MethodsQuantitative real-time polymerase chain reaction, Western blot and immunohistochemistry were respectively performed to detect Rab25 mRNA and protein expression in PCa and adjacent non-cancerous prostate tissues. Receiver-operating characteristic curve analysis was used to evaluate predictive diagnostic value of Rab25. Associations of Rab25 expression with various clinicopathological characteristics and biochemical recurrence-free survival of PCa patients were statistically evaluated. In vitro, PCa cell proliferation was assessed by CCK-8 assay, and the cell migration and invasion activities were evaluated by Transwell assay, following the transfection of Rab25 small interfering RNA.ResultsRas-related protein 25 mRNA and protein expression in PCa tissues were both significantly higher than adjacent non-cancerous prostate tissues (both P < 0.001). The area under the curve of Rab25 immunoreactive score (IRS) was 0.896 (P < 0.001) with 74.0% sensitivity and 95.0% specificity. High Rab25 IRS was significantly associated with high Gleason score (P = 0.02) and distant metastasis (P = 0.01). PCa patients with high Rab25 IRS had shorter overall and biochemical recurrence-free survivals than those with low Rab25 IRS (both P < 0.001). Cox regression analysis identified Rab25 as an independent biomarker for both overall and biochemical recurrence-free survivals of PCa patients. By exploring its activities in vitro, Rab25 downregulation was found to inhibit PCa cell proliferation, migration and invasion.ConclusionsHigh expression of Rab25 may contribute to malignant progression and biochemical recurrence of PCa patients after radical prostatectomy.

Rab7-a novel redox target that modulates inflammatory pain processing.

Chronic pain is accompanied by production of reactive oxygen species (ROS) in various cells that are important for nociceptive processing. Recent data indicate that ROS can trigger specific redox-dependent signaling processes, but the molecular targets of ROS signaling in the nociceptive system remain largely elusive. Here, we performed a proteome screen for pain-dependent redox regulation using an OxICAT approach, thereby identifying the small GTPase Rab7 as a redox-modified target during inflammatory pain in mice. Prevention of Rab7 oxidation by replacement of the redox-sensing thiols modulates its GTPase activity. Immunofluorescence studies revealed Rab7 expression to be enriched in central terminals of sensory neurons. Knockout mice lacking Rab7 in sensory neurons showed normal responses to noxious thermal and mechanical stimuli; however, their pain behavior during inflammatory pain and in response to ROS donors was reduced. The data suggest that redox-dependent changes in Rab7 activity modulate inflammatory pain sensitivity.

TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells.

Recycling endosomes are generally thought to play a central role in endocytic recycling, but recent evidence has indicated that they also participate in other cellular events, including cytokinesis, autophagy, and neurite outgrowth. Rab small GTPases are key regulators in membrane trafficking, and although several Rab isoforms, e.g., Rab11, have been shown to regulate recycling endosomal trafficking, the precise mechanism by which these Rabs regulate recycling endosomes is not fully understood. In this study, we focused on a Rab-GTPase-activating protein (Rab-GAP), one of the key regulators of Rabs, and comprehensively screened 43 mammalian Tre-2/Bub2/Cdc16 (TBC)/Rab-GAP-domain-containing proteins (TBC proteins) for proteins that specifically localize on recycling endosomes in mouse embryonic fibroblasts (MEFs). Four of the 43 mammalian TBC proteins screened, i.e., TBC1D11, TBC1D12, TBC1D14, and EVI5, were found to colocalize well with transferrin receptor, a well-known recycling endosome marker. We further investigated the biochemical properties of TBC1D12, a previously uncharacterized TBC protein. The results showed that TBC1D12 interacted with active Rab11 through its middle region and that it did not display Rab11-GAP activity in vitro. The recycling endosomal localization of TBC1D12 was found to depend on the expression of Rab11. We also found that TBC1D12 expression had no effect on common Rab11-dependent cellular events, e.g., transferrin recycling, in MEFs and that it promoted neurite outgrowth, a specialized Rab11-dependent cellular event, of PC12 cells independently of its GAP activity. These findings indicated that TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells.

Knockdown of Ras-Related Protein 25 (Rab25) Inhibits the In Vitro Cytotoxicity and In Vivo Antitumor Activity of Human Glioblastoma Multiforme Cells.

Ras-related protein 25 (Rab25) is a member of the Rab family, and it has been reported to play an important role in tumorigenesis. However, its direct involvement in human glioblastoma multiforme (GBM) is still unclear. The aim of the current study was to investigate the potential role of Rab25 in the growth, proliferation, invasion, and migration of human GBM. Our results showed that Rab25 expression was significantly higher in human GBM cell lines compared with a normal astrocyte cell line. In vitro functional studies revealed that knockdown of Rab25 reduced cell proliferation and inhibited invasion and migration of GBM cells. In vivo experiments showed that knockdown of Rab25 attenuated the tumor growth in nude mice. Finally, knockdown of Rab25 significantly inhibited the phosphorylation levels of PI3K and AKT in GBM cells. Taken together, these findings indicate that Rab25 may act as a tumor promoter in human GBM and that approaches to target Rab25 may provide a novel strategy to treat this disease.