Expression Of Rab7 Research Articles

Abstract LB-129: Loss of Rab25 expression promotes tumor formation and angiogenesis in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide and is particularly prevalent in some areas of Asian countries. With the advent of next-generation sequencing technologies, it is now possible for us to integrate and analyze large amounts of data at genome-wide scale, with the goal for a better understanding of cellular function and disease development. In order to elucidate the molecular mechanisms that drive tumorigenesis in ESCC, we employed a high-throughput sequencing technology named RNA-Seq (RNA sequencing) to identify dysregulated gene expression in 14 patient-derived ESCC or adjacent non-tumor (NT) clinical specimens of Chinese origin. The expression of Rab25, a family member of Ras-associated binding GTPases, was found to be downregulated in tumour tissues significantly. By both qPCR and immunohistochemistry analysis on a tissue microarray, expression of Rab25 was found to be significantly decreased in a larger cohort of ESCC tumors compared with NT counterparts. Patients with higher levels of Rab25 expression were also significantly associated with a better overall survival. Consistently, absent or downregulated Rab25 expression was also detected in all ESCC cell lines examined as compared to a pooled normal tissue control. Rab25 expression in ESCC cell lines could be restored by demethylation treatment with 5-aza-dC, suggesting Rab25 is silenced via epigenetic alterations. Rab25 functions as an important mediator of vesicle trafficking, regulating cell polarity and distribution of membrane proteins, which are important for the maintenance of normal epithelial lining of the esophagus. We hypothesized that loss of Rab25 expression could lead to dysregulation in the maintenance of this normal epithelium and thus result in tumor development. The tumor suppressive function of Rab25 and the tumorigenic effect of its loss were evaluated by lentiviral-based overexpression and shRNA-knockdown of Rab25 in ESCC cell lines. In vivo studies found Rab25 to suppress tumor formation and angiogenesis in nude mice. Rab25-knockdwon cells gave rise to significantly more and larger tumors as compared to negative control cells. Immunohistochemical staining of resected xenografts revealed an increase in microvessel density and tumor proliferation as shown by higher expressions of endothelial marker CD34 and proliferation marker PCNA, respectively, in Rab25-knockdown cells as compared to negative controls. Similarly, conditioned medium from Rab25-knockdown cells enhanced tube-forming capability of HUVECs, suggesting potential regulation of angiogenic factors by Rab25-mediated pathway. Work on the identification of potential angiogenic factors regulated by Rab25 is now currently underway using a human angiogenesis antibody array. In summary, Rab25 functions as an important tumor suppressor gene in ESCC development by regulating angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-129. doi:10.1158/1538-7445.AM2011-LB-129

NF-κB activation is critical for bacterial lipoprotein tolerance-enhanced bactericidal activity in macrophages during microbial infection (116.15)

Abstract Tolerance to bacterial cell-wall components including gram-positive bacterial lipoprotein (BLP) represents an essential regulatory mechanism during bacterial infection. Our previous work has demonstrated that BLP-induced tolerance, characterised by hyporesponsiveness in producing proinflammatory cytokines and simultaneously enhanced antimicrobial functions, protects against microbial sepsis-related lethality. However, the underlying mechanisms remain unidentified. In this study we reported that upon S. aureus or E. coli challenge, significantly enhanced IκBα phosphorylation and p65 translocation into the nucleus were observed in BLP-tolerised bone marrow-derived macrophages (BMM), indicating that NF-κB is activated in BLP-tolerised macrophages during bacteria infection. To further clarify whether activation of the NF-κB pathway is required for efficient bacterial killing by BLP-tolerised macrophages, we used two NF-κB inhibitors, SN50 and SC-154. Both inhibitors substantially restrained BLP-tolerised macrophage-induced intracellular bacterial killing, and this was closely associated with delayed phagolysosome fusion. Furthermore, the expression of LAMP-1 and Rab5, two membrane-trafficking regulators participated the process of phagosome maturation was significantly downregulated when the NF-κB pathway was blocked. Collectively, our results highlight a novel role of the NF-κB pathway in bactericidal activity displayed by BLP-tolerised macrophages during microbial infection.

MicroRNA-451 functions as a tumor suppressor in human non-small cell lung cancer by targeting ras-related protein 14 (RAB14)

Accumulating evidence suggests that microRNAs (miRNAs) are important gene regulators, which can have critical roles in diverse biological processes including tumorigenesis. In this study, we analyzed the miRNA expression profiles in non-small cell lung carcinoma (NSCLC) by use of a miRNA microarray platform and identified 40 differentially expressed miRNAs. We showed that miRNA (miR)-451 was the most downregulated in NSCLC tissues. The expression level of miR-451 was found to be significantly correlated with tumor differentiation, pathological stage and lymph-node metastasis. Moreover, low miR-451 expression level was also correlated with shorter overall survival of NSCLC patients (P<0.001). Ectopic miR-451 expression significantly suppressed the in vitro proliferation and colony formation of NSCLC cells and the development of tumors in nude mice by enhancing apoptosis, which might be associated with inactivation of Akt signaling pathway. Interestingly, ectopic miR-451 expression could significantly inhibit RAB14 protein expression and decrease a luciferase-reporter activity containing the RAB14 3'-untranslated region (UTR). In addition,, RNA interference silencing of RAB14 gene could recapitulate the tumor suppressor function of miR-451, whereas restoration of RAB14 expression could partially attenuate the tumor suppressor function of miR-451 in NSCLC cells. Furthermore, we also showed that strong positive immunoreactivity of RAB14 protein was significantly associated with downregulation of miR-451 (P=0.01). These findings suggest that miR-451 regulates survival of NSCLC cells partially through the downregulation of RAB14. Therefore, targeting with the miR-451/RAB14 interaction might serve as a novel therapeutic application to treat NSCLC patients.

Pulmonary Surfactant Protein A Enhances Endolysosomal Trafficking in Alveolar Macrophages through Regulation of Rab7

Surfactant protein A (SP-A), the most abundant pulmonary soluble collectin, modulates innate and adaptive immunity of the lung, partially via its direct effects on alveolar macrophages (AM), the most predominant intra-alveolar cells under physiological conditions. Enhanced phagocytosis and endocytosis are key functional consequences of AM/SP-A interaction, suggesting a SP-A-mediated modulation of small Rab (Ras related in brain) GTPases that are pivotal membrane organizers in both processes. In this article, we show that SP-A specifically and transiently enhances the protein expression of endogenous Rab7 and Rab7b, but not Rab5 and Rab11, in primary AM from rats and mice. SP-A-enhanced GTPases are functionally active as determined by increased interaction of Rab7 with its downstream effector Rab7 interacting lysosomal protein (RILP) and enhanced maturation of cathepsin-D, a function of Rab7b. In AM and RAW264.7 macrophages, the SP-A-enhanced lysosomal delivery of GFP-Escherichia coli is abolished by the inhibition of Rab7 and Rab7 small interfering RNA transfection, respectively. The constitutive expression of Rab7 in AM from SP-A(-/-) mice is significantly reduced compared with SP-A(+/+) mice and is restored by SP-A. Rab7 blocking peptides antagonize SP-A-rescued lysosomal delivery of GFP-E. coli in AM from SP-A(-/-) mice. Activation of Rab7, but not Rab7b, by SP-A depends on the PI3K/Akt/protein kinase Cζ (PKCζ) signal transduction pathway in AM and RAW264.7 macrophages. SP-A induces a Rab7/PKCζ interaction in these cells, and the disruption of PKCζ by small interfering RNA knockdown abolishes the effect of SP-A on Rab7. The data demonstrate a novel role for SP-A in modulating endolysosomal trafficking via Rab7 in primary AM and define biochemical pathways involved.

A role for Rab10 in von Willebrand factor release discovered by an AP‐1 interactor screen in C. elegans

Endothelial von Willebrand factor (VWF) mediates platelet adhesion and acts as a protective chaperone to clotting factor VIII. Rapid release of highly multimerized VWF is particularly effective in promoting hemostasis. To produce this protein, an elaborate biogenesis is required, culminating at the trans-Golgi network (TGN) in storage within secretory granules called Weibel-Palade bodies (WPB). Failure to correctly form these organelles can lead to uncontrolled secretion of low-molecular-weight multimers of VWF. The TGN-associated adaptor AP-1 and its interactors clathrin, aftiphilin and γ-synergin are essential to initial WPB formation at the Golgi apparatus, and thus to VWF storage and secretion. To identify new proteins implicated in VWF storage and/or secretion. A genomewide RNA interference (RNAi) screen was performed in the Nematode C. elegans to identify new AP-1 genetic interactors. The small GTPase Rab10 was found to genetically interact with a partial loss of function of AP-1 in C. elegans. We investigated Rab10 in human primary umbilical vein endothelial cells (HUVECs). We report that Rab10 is enriched at the Golgi apparatus, where WPB are formed, and that in cells where Rab10 expression has been suppressed by siRNA, VWF secretion is altered: the amount of rapidly released VWF was significantly reduced. We also found that Rab8A has a similar function. Rab10 and Rab8A are new cytoplasmic factors implicated in WPB biogenesis that play a role in generating granules that can rapidly respond to secretagogue.

Metformin induces Rab4 through AMPK and modulates GLUT4 translocation in skeletal muscle cells

Metformin is a major oral anti-diabetic drug and is known as an insulin sensitizer. However, the mechanism by which metformin acts is unclear. In this study, we found that AICAR, an AMPK activator, and metformin increased the expression of Rab4 mRNA and protein levels in skeletal muscle C2C12 cells. The promoter activity of Rab4 was increased by metformin in an AMPK-dependent manner. Metformin stimulated the phosphorylation of AS160, Akt substrate, and Rab GTPase activating protein (GAP), and also increased the phosphorylation of PKC-zeta, which is a critical molecule for glucose uptake. Knockdown of AMPK blocked the metformin-induced phosphorylation of AS160/PKC-zeta. In addition, a colorimetric absorbance assay showed that insulin-induced translocation of GLUT4 was suppressed in Rab4 knockdown cells. Moreover, Rab4 interacted with PKC-zeta but not with GLUT4. The C-terminal-deleted Rab4 mutant, Rab4ΔCT, showed diffuse sub-cellular localization, while wild-type Rab4 localized exclusively to the perinuclear membrane. Unlike Rab4ΔCT, wild-type Rab4 co-localized with PKC-zeta. Together, these results demonstrate that metformin induces Rab4 expression via AMPK-AS160-PKC-zeta and modulates insulin-mediated GLUT4 translocation.

Transformation of rat intestinal epithelial cells by overexpression of Rab25 is microtubule dependent

Little research has addressed the role of membrane trafficking and recycling in the regulation of the transformed phenotype of neoplastic cells. The small GTPase Rab25 is an epithelial-specific modulator of membrane recycling. Recent studies have demonstrated that Rab25 expression is up-regulated in a number of epithelial cancers and overexpression may increase the aggressive phenotype of certain cancers. We have utilized the nontransformed RIE cell line to examine the influence of Rab25 on transformation. Overexpression of Rab25 in RIE cells leads to morphological transformation as well as growth in soft agar, tumor formation in nude mice, disruption of integrin-based focal adhesions, and alteration in modified microtubule subsets. Although the predominance of recent cancer research has focused on the manipulation of the actin-based cytoskeleton, recycling trafficking relies on microtubules. Transformation of RIE cells through overexpression of Rab25, but not with H-Ras(V12) , was reversed by inhibitors of microtubule polymerization. These results suggest that up-regulation of Rab25 in RIE cells leads to microtubule-dependent transformation. Thus, depolymerization of microtubules may be a potent therapeutic target for cancer therapy through the reversal of the invasive phenotype of certain cancer cells.

A CRE that binds CREB and contributes to PKA-dependent regulation of the proximal promoter of human RAB25 gene

RAB25 plays an important role in tumor progression and aggressiveness; altered RAB25 expression may cause human cancer. As the underlying mechanism of RAB25-mediated carcinogenesis in various tumor types progressively comes to light, RAB25 is expected to represent a novel therapeutic target. However, the regulation of RAB25 expression per se has not yet been described. Here we have firstly identified and characterized the human RAB25 promoter. Using PCR-based chromatin accessibility and chromatin immunoprecipitation (ChIP) assays, an open chromatin conformation (−173/+17) was detected around the transcription start site of the RAB25 gene. Deletion constructs of the 5′ flanking region were fused to a luciferase reporter gene. After transient transfection in gastric cancer cell line AGS, a CRE (−67/−58) binding CREB was identified in the core promoter region. Electrophoretic mobility shift (EMSA) and ChIP assays demonstrated that CREB binds to the core promoter. Deletion of CREB consensus sequence resulted in the total loss of the promoter activity. Moreover, we have also found forskolin, PKA activator, could enhance open chromatin accessibility, by which to expose the CRE and facilitate phosphorylation of CREB, which in turn recruits co-factor CBP and Brg I and then results in a more open chromatin configuration associated with local histone modification, finally heightening RAB25 expression and strengthening its promoter activity. Therefore, the present study delineates the fundamental elements of a core promoter structure that will be helpful for future studies regarding the regulation of RAB25 gene.

Rab25 as a tumour suppressor in colon carcinogenesis

Recent investigations have increasingly focussed attention on the roles of intracellular vesicle trafficking in the regulation of epithelial polarity and transformation. Rab25, an epithelial-specific member of the Rab family of small GTPases, has been associated with several epithelial cancers. Whereas Rab25 overexpression is associated with ovarian cancer aggressive behaviour, Rab25 expression is decreased in human colon cancers independent of stage. Recent studies of mouse models of intestinal and colonic neoplasia have demonstrated that Rab25 deficiency markedly promotes the development of neoplasia. Some of these effects appear related to alterations in β1-integrin trafficking to the cell surface. These findings all suggest that Rab25 is a tumour suppressor for colonic neoplasia.

Deacetylation of FoxO by Sirt1 Plays an Essential Role in Mediating Starvation-Induced Autophagy in Cardiac Myocytes

autophagy, a bulk degradation process of cytosolic proteins and organelles, is protective during nutrient starvation in cardiomyocytes (CMs). However, the underlying signaling mechanism mediating autophagy is not well understood. we investigated the role of FoxOs and its posttranslational modification in mediating starvation-induced autophagy. glucose deprivation (GD) increased autophagic flux in cultured CMs, as evidenced by increased mRFP-GFP-LC3 puncta and decreases in p62, which was accompanied by upregulation of Sirt1 and FoxO1. Overexpression of either Sirt1 or FoxO1 was sufficient for inducing autophagic flux, whereas both Sirt1 and FoxO1 were required for GD-induced autophagy. GD increased deacetylation of FoxO1, and Sirt1 was required for GD-induced deacetylation of FoxO1. Overexpression of FoxO1(3A/LXXAA), which cannot interact with Sirt1, or p300, a histone acetylase, increased acetylation of FoxO1 and inhibited GD-induced autophagy. FoxO1 increased expression of Rab7, a small GTP-binding protein that mediates late autophagosome-lysosome fusion, which was both necessary and sufficient for mediating FoxO1-induced increases in autophagic flux. Although cardiac function was maintained in control mice after 48 hours of food starvation, it was significantly deteriorated in mice with cardiac-specific overexpression of FoxO1(3A/LXXAA), those with cardiac-specific homozygous deletion of FoxO1 (c-FoxO1(-/-)), and beclin1(+/-) mice, in which autophagy is significantly inhibited. these results suggest that Sirt1-mediated deacetylation of FoxO1 and upregulation of Rab7 play an important role in mediating starvation-induced increases in autophagic flux, which in turn plays an essential role in maintaining left ventricular function during starvation.

Alteraciones en el reclutamiento y activación de proteínas Rab durante la infección micobacteriana

At the phagosome level, Mycobacterium spp. alters activation and recruitment of several "Ras gene from rat brain" proteins, commonly known as Rab. Mycobacterial phagosomes have a greater and sustained expression of Rab5, Rab11, Rab14 and Rab22a, and lowered or no expression of Rab7, Rab9 and Rab6. This correlates with increased fusion of the phagosomes with early and recycling endosomes acquiring some features of early phagosomes, allowing the bacteria to gain access to nutrients and preventing the activation of anti-mycobacterial mechanisms. The expression of constitutively active mutants of Rab from the early stage endosomes prevents the maturation of phagosomes containing latex beads or heat-inactivated mycobacteria. Silencing of these mutants by interference RNA or dominant negative forms induces the maturation of mycobacterial phagosomes. The mechanisms have not been established by which mycobacteria alter the expression of these GTPases and thereby shift the phagolysosomal maturation. The problem can be explained by alterations in the recruitment of proteins that interact with Rab, such as phosphoinositide 3-kinases and early endosomal antigen 1. Identifying the mechanisms used by Mycobacterium spp. to disrupt the cycle of Rab activation will be essential to understand the pathophysiology of mycobacterial infections and usefully to potential drug targets.

Ras-related protein Rab10 facilitates TLR4 signaling by promoting replenishment of TLR4 onto the plasma membrane

The Toll-like receptor (TLR)4 receptor complex, TLR4/MD-2, plays an important role in the inflammatory response against lipopolysaccharide, a ubiquitous membrane component in Gram-negative bacteria. Ligand recognition by TLR4 initiates multiple intracellular signaling pathways, leading to production of proinflammatory mediators and type I IFN. Ligand interaction also leads to internalization of the surface receptor complex into lysosomes, leading to the degradation of TLR4 and the termination of LPS response. However, surface level of TLR4 receptor complex is maintained via continuous replenishment of TLR4 from intracellular compartments like Golgi and endosomes. Here we show that continuous replenishment of TLR4 from Golgi to plasma membrane is regulated by the small GTPase Rab10, which is essential for optimal macrophage activation following LPS stimulation. Expression of Rab10 is inducible by LPS. Blockade of Rab10 function leads to decreased membrane TLR4 expression and diminished production of inflammatory cytokines and interferons upon LPS stimulation. These findings suggest that Rab10 expression provides a mechanism to refine TLR4 signaling by regulating the trafficking rate of TLR4 onto the plasma membrane. In addition, we show that altered Rab10 expression in macrophages influences disease severity in an in vivo model of LPS-induced acute lung injury, suggesting Rab10 as a possible therapeutic target for human acute respiratory distress syndrome (ARDS).

Specific Behavior of Intracellular Streptococcus pyogenes That Has Undergone Autophagic Degradation Is Associated with Bacterial Streptolysin O and Host Small G Proteins Rab5 and Rab7

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLO-deficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells.

Rab11 and Actin Cytoskeleton Participate in Giardia lamblia Encystation, Guiding the Specific Vesicles to the Cyst Wall

Background Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins.Methodology and Principal FindingsIn this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol.Conclusions and SignificanceOur results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and actin may be useful as novel pharmacological targets.

CMT2B-associated Rab7 mutants inhibit neurite outgrowth

Charco-Marie-Tooth type 2B (CMT2B) neuropathy is a rare autosomal-dominant axonal disorder characterized by distal weakness, muscle atrophy, and prominent sensory loss often complicated by foot ulcerations. CMT2B is associated with mutations of the Rab7 protein, a small GTPase controlling late endocytic traffic. Currently, it is still unknown how these mutations cause the neuropathy. Indeed, CMT2B selectively affects neuronal processes, despite the ubiquitous expression of Rab7. Therefore, this study focused on whether these disorder-associated mutations exert an effect on neurite outgrowth. We observed a marked inhibition of neurite outgrowth upon expression of all the CMT2B-associated mutants in the PC12 and Neuro2A cell lines. Thus, our data strongly support previous genetic data which proposed that these Rab7 mutations are indeed causally related to CMT2B. Inhibition of neurite outgrowth by these CMT2B-associated Rab7 mutants was confirmed biochemically by impaired up-regulation of growth-associated protein 43 (GAP43) in PC12 cells and of the nuclear neuronal differentiation marker NeuN in Neuro2A cells. Expression of a constitutively active Rab7 mutant had a similar effect to the expression of the CMT2B-associated Rab7 mutants. The active behavior of these CMT2B-associated mutants is in line with their previously demonstrated increased GTP loading, thus confirming that active Rab7 mutants are responsible for CMT2B. Our findings provide an explanation for the ability of CMT2B-associated Rab7 mutants to override the activity of wild-type Rab7 in heterozygous patients. Thus, our data suggest that lowering the activity of Rab7 in neurons could be a targeted therapy for CMT2B.

Abstract 3666: Follicle-stimulating hormone inhibits apoptosis and autophagy in ovarian cancer cells by activating survivin and Rab25

Abstract Follicle-stimulating hormone (FSH) has been demonstrated to increase the risk of ovarian malignancy, but the mechanisms remains unclear. Our previous results showed that FSH increases the expression of VEGF through activation of survivin, which is mediated by the activation of the PI3K/AKT signaling pathway. We reported, herein, that FSH promoted ovarian cancer cells proliferation and prevented apoptosis in vitro and in vivo. FSH up-regulated the expression of survivin and down-regulated the expression of programmed cell death 6 (PDCD6) and death receptor 5 (DR5), two molecules required for apoptosis induction. Further, we have defined that the decreased expression of DR5, but not PDCD6, was mediated by survivin. We have established animal xenografts tissue arrays, the tumors from the groups with FSH treatment had higher expression of survivin and lower expression of DR5 comparing with the tumors from untreated groups, further confirming the interaction of FSH-survivin-DR5. In addition, we have demonstrated that FSH does not only inhibit apoptosis, but also inhibit autophagy. FSH inhibits autophagy by activating mTOR pathway in ovarian cancer cells under different stress conditions, including serum starvation. By Reverse Phase Protein Arrays (RPPA) analysis and western blot confirmation, we found that FSH stimulation increases the level of Rab25 in multiple ovarian cancer cells in a dose dependent manner. Rab25, a small GTPase, is found to be associated with the progression of ovarian tumor. Rab25, belongs to Rab11 supfamily, was shown to involve in endosomal recycling. To study the role of FSH-Rab25 interaction in regulating autophagy, we knocked down Rab25 expression in ES-2, Hey and SKOv3 ovarian cancer cell lines by siRNA, and examined the effects of FSH on autophagy. Rab25 knockdown increased the starvation induced autophagy through the inhibition of pAkt, while FSH administration could partly reverse the effects of Rab25 knockdown, due to the increase of Rab25 expression stimulated by FSH. Our findings indicated that Rab25 is one of the molecules regulated by FSH in ovarian cancer cells that has a novel role as a regulator in autophagy. Taken together, we have demonstrated that FSH promotes ovarian cancer development through regulating expression of survivin, Rab25, PDCD6 and DR5, which in turn regulates cell proliferation, apoptosis as well as autophagy. Further understanding the molecular mechanisms of FSH in the process of ovarian carcinogenesis and development will ultimately help developing an effective cancer prevention method, and find a novel targeted therapy for ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3666.

Abstract 3073: Rab25 expression, loss and therapeutic implications in human breast cancer

Abstract Introduction: Our previous work has shown that the loss of Rab25 occurs in estrogen receptor (ER) and progesterone receptor (PR) negative breast cancer cell lines and human tumor samples and seems to correlate with an active ras pathway. We have expressed Rab25 in several hormonally insensitive breast cancer cell lines and have demonstrated marked reduction in tumor growth. Histologically, the tumor cells appear smaller, and angiogenesis is significantly reduced. We have investigated the mechanism by which Rab25 exerts its anti-tumorigenic effect. Loss of Rab25 cooperates with mutant Ras to promote tumorigenesis in primary human mammary epithelial cells (HMEC). HIF1-alpha levels are markedly increased with loss of Rab25 if Ras is overexpressed. Rab25 is expressed in both luminal and basal mammary epithelium at similar levels. The loss of rab25 enhances VEGF-A secretion and VEGFR1 expression. Monoclonal antibodies can be potentially used to target these pathways which appear to be active in Rab25 negative tumors. Materials and Methods: Cell culture - MDA-MB-231 cells were grown in DME medium with 10% FBS, and Rao-3 cell line and HMEC were grown in DFCI-1 medium. Cells were transduced with retroviral supernatant and selected. Expression of RAB25 in transduced cells was determined by RT-PCR and western blot. Silencing of Rab25-Rab-25 was silenced in RAO-2 cells by pENTRTM/H1/TO siRNA system (Invitrogen, Carlsbad, CA). Our shRNA sequences are designed using the BLOCK-iTTM RNAi Designer. The most effective silencer of Rab25 was selected on the basis of testing 3 sequence pairs. The double-stranded oligos were inserted into the pENTR/H1/TO vector and transfected into cells. Silencing of Rab25 in transduced cells was confirmed by RT-PCR and Western blot. qPCR analysis- Expression of HIF1-alpha was quantified using real-time PCR with fluorescence detection. Western blot- Western blot was performed on cell extracts for rab25 and ras. Immunohistochemistry - CK5/6 served as a basal maker, and CK18 served as a luminal maker for co-staining with Rab25 in human mammary gland. In vivo tumor formation assay − 5 × 106 cells in 0.1 ml PBS were injected into the left mammary fat pad of five week-old female nude mice. Mice were monitored for the following 7 weeks for tumor volume. Therapeutic assays- Cell lines were incubated with anti-VEGF-A, anti-VEGFR1, or both, and cell numbers were tabulated over 5 days. Results: 1) Rab25 loss cooperated with Ras to promote tumorigenesis in several donors. 2) HIF1α levels rose significantly with loss of Rab25 and Ras co-expression. 3) Rab25 is expressed in both basal and luminal normal mammary epithelium. 4) Both anti-VEGF-A and anti-VEGFR1 antibodies have therapeutic implications for Rab25 negative breast cancer cell lines. Conclusions: Rab25 is a tumor suppressor that antagonizes Ras and may be part of an important therapeutic pathway. Its loss may serve as therapeutic marker for certain types of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3073.

Differential role of Rab proteins in ciliary trafficking: Rab23 regulates Smoothened levels

The structure and function of the primary cilium as a sensory organelle depends on a motor-protein-powered intraflagellar transport system (IFT); defective IFT results in retinal degeneration and pleiotropic disorders such as the Bardet Biedl syndrome (BBS) and defective hedgehog (HH) signaling. Protein transport to the cilium involves Rab GTPases. Rab8, together with a multi protein complex of BBS proteins, recruits cargo to the basal body for transport to the cilium. Loss of Rab23 in mice recapitulates the HH phenotype but its function in HH signaling is unknown. Here we established a novel protocol, based on fluorescence recovery after photo-bleaching (FRAP), allowing the quantitative analysis of protein transport into the cilium of MDCK cells. We compared the effect of Rab8, Rab5 and Rab23 on the ciliary transport of the HH-associated transmembrane receptor Smoothened, the microtubular tip protein EB1, and the receptor protein Kim1. Ciliary FRAP confirmed the role of Rab8 in protein entry to the cilium. Dominant negative Rab5 had no impact on the ciliary transport of Smoothened or EB1, but slowed the recovery of the apical protein Kim1 in the cilium. Depletion of Rab23 or expression of dominant-negative Rab23 decreased the ciliary steady state specifically of Smoothened but not EB1 or Kim1, suggesting a role of Rab23 in protein turnover in the cilium.

Activation of the nitric oxide-mitochondrial hyperpolarization-mTOR-Rab4 signaling pathway precedes disease development in lupus T cells (93.36)

Abstract Nitric oxide (NO) dependent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T cell activation in Systemic Lupus Erythematosus. Activity of mTOR, which senses the mitochondrial membrane potential, is increased in lupus T cells, which promotes HRES-1/Rab4 expression. Rab4 overexpression enhances recycling and lysosomal degradation of immune synapse components, including CD4 and TCRζ. We investigated activity of the NO-MHP-mTOR-Rab4 signaling pathway in lupus-prone mice. Overexpression of Rab4 and loss of TCRζ were detected prior to disease onset of NZB/NZW (F1) mice at 5 months. Increased NO production, mitochondrial transmembrane potential, and mitochondrial mass were present after disease onset at 11 months. MRL/lpr mice exhibited increased expression of the mitochondrial voltage-dependent anion channel 1(VDAC1) protein, transaldolase (TAL), Rab4, mTOR activation, and enhanced CD3 and CD4 recycling at 2-3 months, prior to disease development. In conclusion, lupus-prone mice exhibit mitochondrial dysfunction, with increased NO production, MHP, elevated expression of VDAC1 and TAL, and mTOR activation. Increased Rab4 expression was associated with enhanced endocytic recycling of CD4 and CD3. The results suggest that activation of the NO-MHP-mTOR-Rab4 signaling pathway is detectable and precedes disease onset in lupus-prone mice.

Involvement of Rab9 and Rab11 in the intracellular trafficking of TRPC6

TRPC proteins become involved in Ca2+ entry following the activation of Gq-protein coupled receptors. TRPC6 is inserted into the plasma membrane upon stimulation and remains in the plasma membrane as long as the stimulus is present. However, the mechanism that regulates the trafficking of TRPC6 is unclear. In the present study, we highlighted the involvement of two Rab GTPases in the trafficking of TRPC6. Rab9 co-localized in vesicular structures with TRPC6 in HeLa cells and co-immunoprecipitated with TRPC6. When co-expressed with TRPC6, Rab9S21N, a dominant negative mutant, caused an increase in the level of TRPC6 at the plasma membrane and in TRPC6-mediated Ca2+ entry upon activation by a muscarinic receptor agonist. Similarly, the expression of Rab11 also caused an increase in TRPC6 expression at the cell surface and an increase in TRPC6-mediated Ca2+ entry. The co-expression of TRPC6 with the dominant negative mutant Rab11S25N abolished CCh-induced TRPC6 activation and reduced the level of TRPC6 at the plasma membrane. This study demonstrates that the trans-Golgi network and recycling endosomes are involved in the intracellular trafficking of TRPC6 by regulating channel density at the cell surface.