Rab3 proteins and cancer: Exit strategies.

Rab proteins are GTPases involved in all stages of vesicular transport and membrane fusion in mammalian cells. Individual Rab proteins localize to specific cellular organelles and regulate a specific membrane trafficking pathway. Recent studies suggest an important role for Rab proteins in cancer. Rab3 isoforms (Rab3A, Rab3B, Rab3C, and Rab3D) are expressed almost exclusively in neurons and secretory cells. In this review, the role of Rab3 isoforms in a variety of tumor types is discussed. Of the four Rab3 isoforms, Rab3D has been studied most extensively in cancer cells and this isoform appears to play an oncogenic role in breast, colon, esophageal, skin, and brain tumors. Overexpression of Rab3A and Rab3C was observed in gliomas and colon cancers, respectively. Increased expression of the Rab3 isoforms is related to increased proliferation, migration, and invasiveness. Moreover, high Rab3 isoform levels are often associated with decreased survival and advanced pathological stage in clinical samples. Rab3 isoform-dependent activation of the AKT pathway has been observed in several studies. Although the effects of Rab3 isoforms on cancer cell growth and function have been examined in many tumor types, a number of important questions remain. Are the Rab3-positive vesicles in cancer cells actually secretory in nature? If so, are the contents of these vesicles secreted in a regulated or constitutive manner? How does Rab3-regulated secretion affect cellular signaling and tumor growth? Finally, can Rab3 isoforms be therapeutically manipulated in cancer cells? The fact that knockout of a single Rab3 isoform does not affect viability, at least in mouse models, suggests that targeting of these proteins may be a safe and effective treatment strategy for tumor cells expressing any of the Rab3 isoforms.

Chibby is a weak regulator of ß-catenin activity in gastric epithelium.

The canonical Wnt-ß-catenin pathway is important in normal development. Mutations in ß-catenin or proteins involved with regulating its phosphorylation or localization result in its nuclear accumulation where it activates its target genes and stimulates cell proliferation. This pathway is dysregulated in many different types of cancer, including gastric cancer (GC). Chibby (Cby) is a 14-kDa protein that inhibits ß-catenin localization to the nucleus and represses ß-catenin-induced transcriptional activity. In the current study, we examined the expression and function of Cby in normal and cancerous human gastric tissue. Reverse-transcription polymerase chain reaction and immunohistochemistry revealed that Cby is expressed in human stomach and localized to glandular elements. Immunohistochemical staining intensity of Cby was decreased in GC tissue when compared with normal gastric epithelium. In AGS cells, a human gastric carcinoma cell line, Cby expression was low. Stable AGS cell transfectants overexpressing Cby were prepared. Cby overexpression did not affect proliferation rates or ß-catenin levels. However, confocal microscopy and subcellular fractionation studies revealed that Cby overexpression resulted in a small decrease in nuclear ß-catenin. Moreover, Cby overexpression caused a molecular weight shift in nuclear ß-catenin and resulted in decreased ß-catenin signaling in AGS cells as measured by the TopFlash assay. However, Cby overexpression did not affect c-Myc protein levels. To conclude, Cby expression was decreased in GC samples and Cby expression altered ß-catenin localization in cultured GC cells. However, Cby did not affect cell proliferation rates or ß-catenin-induced protein expression. Cby may be involved in the early events in the pathogenesis of GC.

Variations in the multimerization region of the Helicobacter pylori cytotoxin CagA affect virulence.

Helicobacter pylori colonizes the human stomach by infecting gastric epithelial cells. It is the primary cause of peptic ulcer disease and gastric cancer (GC). Cytotoxin-associated gene A (CagA) is a virulence factor produced by H. pylori. Strains positive for the CagA protein are associated with more severe gastric diseases. The 3' region of the cagA gene exhibits heterogeneity with respect to tyrosine phosphorylation motifs (EPIYA) and CagA multimerization motifs (CM). CagA proteins are categorized as either Western or Eastern based on EPIYA sequences. CM motifs are also identified as Western and Eastern based on CM sequences identified in Western and East Asian countries. It has been suggested that CagA proteins possessing an Eastern CM type are associated with less severe gastric disorders. In the present study, the effects of two CagA peptides with different CM motifs on cell function were compared: CagA with a Western and Eastern CM motif (CagA-WE), and CagA with two Western CM motifs (CagA-WW). CagA sequences were fused with green fluorescent protein (GFP) to form GFP-CagA fusion proteins. GFP-CagA and GFP control constructs were transfected into human gastric adenocarcinoma cells (AGS). GFP-CagA expression was verified by immunoblotting and immunofluorescence. The results demonstrated that, following 18 h, the CagA-WE-transfected cells were less adherent compared with the CagA-WW transfected cells. CagA has also been reported to cause cell elongation in AGS cells. In the current study, cell elongation was more frequent in the CagA-WW-transfected cells compared with the CagA-WE transfected cells (8.34 vs. 3.97% cells, respectively). The CagA peptides did not affect proliferation or apoptosis rates. These results suggest that different CM motif types may affect CagA virulence.

Analysis of the 3'-variable region of the cagA gene from Helicobacter pylori strains infecting patients at New York City hospitals.

Helicobacter pylori infects the gastric mucosa in humans and is a causative agent for peptic ulcer disease (PUD) and gastric cancer (GC). CagA is produced by H. pylori and is associated with more severe outcomes. cagA genes vary at the 3'-region with respect to phosphorylation motifs (EPIYA-A, -B, -C, or -D) and CagA multimerization motifs (CM). This variability may be associated with the clinical outcomes. We examined the variable region of cagA genes expressed in H. pylori-infected patients treated at three NYC Hospitals. DNA was isolated from gastric biopsies of patients undergoing upper endoscopy. Most H. pylori-infected patients were Black or Hispanic. The cagA 3'-region of CagA-positive samples was amplified by PCR, purified and sequenced. The patterns of EPIYA and CM motifs were examined and related to clinical outcomes. We obtained 42 CagA sequences from our sample collection. The EPIYA phosphorylation motif pattern was ABC in 81.0% of our samples. Western (W) and Eastern (E) CM motifs have also been defined. CagA proteins lacking an Eastern CM motif and possessing one or two Western CM motifs were observed more frequently in patients with PUD and GC when compared with non-ulcer gastritis (50.0% vs 11.8%, respectively), suggesting that these CM motif patterns are more virulent than those containing at least one Eastern CM motif. We conclude that In H. pylori-infected patients treated at NYC Hospitals, CM motif patterns in the CagA 30-variable region may be more significant than EPIYA motif patterns with respect to clinical outcomes.

Rab3D regulates amylase levels, not agonist-induced amylase release, in AR42J cells.

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.

Hsp90 Co-localizes with Rab-GDI-1 and regulates agonist-induced amylase release in AR42J cells.

Rab proteins are small GTPases required for vesicle trafficking through the secretory and endocytic pathways. Rab GDP-dissociation inhibitor (rab-GDI) regulates Rab protein function and localization by maintaining Rab proteins in the GDP-bound conformation. Two isoforms of rab-GDI are present in most mammalian cells: GDI-1 and GDI-2. It has recently been demonstrated that a Heat shock protein 90 (Hsp90) chaperone complex regulates the interactions between Rab proteins and Rab-GDI-1. The AR42J cell line is derived from rat pancreatic exocrine tumor cells and develops an acinar-like phenotype when treated with dexamethasone (Dex). The aim of the present study was to examine the expression of rab-GDI isoforms and Hsp90 in AR42J cells in the presence or absence of Dex. Rab-GDI:Hsp90 interactions were also examined. Both rab-GDI isoforms were detected in AR42J cells by immunoblotting. In Dex-treated cells, quantitative immunoblotting revealed that rab-GDI-1 expression increased by 28%, although this change was not statistically significant. Rab-GDI-2 levels were unaltered by Dex treatment. Approximately 21% rab-GDI-1 was membrane associated, whereas rab-GDI-2 was exclusively cytosolic. Dex treatment did not affect the subcellular distribution of rab-GDI isoforms. Hsp90 was present in the cytosolic and membrane fractions of AR42J cells and co-immunoprecipitated with cytosolic rab-GDI-1. Moreover, density gradient centrifugation of AR42J cell membranes revealed that Hsp90 and rab-GDI-1 co-localize on low- and high-density membrane fractions, including amylase-containing secretory granules. The Hsp90 inhibitor, geldanamycin, inhibited CCK-8-induced amylase release from these cells in a dose-dependent manner. Our results indicate that as AR42J cells differentiate into acinar-like cells, rab-GDI isoform expression and localization is not significantly altered. Moreover, our findings suggest that Hsp90 regulates agonist-induced secretion in exocrine cells by interacting with rab-GDI-1.

Rab3D redistribution and function in rat parotid acini.

Rab3D is a low molecular weight GTP-binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane-associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co-localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc-tagged Rab3D and Rab3DQ81L, a GTP-binding mutant, were prepared and incubated with streptolysin O-permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D-binding to parotid membranes is guanine nucleotide-dependent. Moreover, wild-type and mutant Rab3D inhibited agonist-induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild-type Rab3D and the GTP-binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition.