RAB2A controls MT1‐MMP endocytic and E‐cadherin polarized Golgi trafficking to promote invasive breast cancer programs

The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER‐to‐Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post‐endocytic trafficking of membrane‐bound MT1‐MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E‐cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination.     

Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia

Remodeling of the extracellular matrix by carcinoma cells during metastatic dissemination requires formation of actin-based protrusions of the plasma membrane called invadopodia, where the trans-membrane type 1 matrix metalloproteinase (MT1-MMP) accumulates. Here, we describe an interaction between the exocyst complex and the endosomal Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) on MT1-MMP–containing late endosomes in invasive breast carcinoma cells. We found that WASH and exocyst are required for matrix degradation by an exocytic mechanism that involves tubular connections between MT1-MMP–positive late endosomes and the plasma membrane in contact with the matrix. This ensures focal delivery of MT1-MMP and supports pericellular matrix degradation and tumor cell invasion into different pathologically relevant matrix environments. Our data suggest a general mechanism used by tumor cells to breach the basement membrane and for invasive migration through fibrous collagen-enriched tissues surrounding the tumor.     

RAB5A基因表达改变对人肺腺癌细胞系GLC-82和SPC-a1的分化和侵袭特性影响

为探讨RAB5A基因对两种人肺腺癌细胞系GLC－82和SPC－al分化及侵袭特性的影响。利用细胞转染技术将构建的RAB5A反义RNA重组质粒（pcDNA3-AntiRAB5A)和RAB5A正义真核表达载体分别转染入低分化人肺腺癌细胞系GLC－82和低转移人肺腺癌细胞系SPC－al中,在稳定筛选后,通过裸鼠体内实验和体外人工基底膜侵袭和细胞趋化运动实验,观察观察转染后细胞分化和转移特性的改变,观察转染前后细胞,发现转染后GLC－82细胞体外侵袭重组基底膜能力及趋化运动能力降低（t检验P＜0．02）;裸鼠体内成瘤实验,瘤块切片病理观察转染后GLC－82细胞出现腺腔样及基底模样结构,分化程度增高,转染后SPC－al细胞体外趋化运动能力,侵袭重组基底膜能力均增强(t检验P＜0．02）。RAB5A基因通过影响细胞的体外趋化运动能力,侵袭重组基底膜能力等对GLC－82和SPC－al细胞的侵袭表型形成及GLC－82细胞的分性发挥重要作用。     

miR-203 Acts as a Tumor Suppressor Gene in Osteosarcoma by Regulating RAB22A

microRNAs (miRNAs), small noncoding RNAs of 19–25 nt, play an important roles in the pathological processes of tumorigenesis. The object of this study was to study the expression and function of miR-203 and to found its target gene in osteosarcoma. In our study, we found the expression level of miR-203 was significantly downregulated in osteosarcoma cell lines and tissues. In addition, overexpression of miR-203 inhibited the osteosarcoma cell proliferation and migration and inhibited Mesenchymal-to-Epithelial reversion Transition (MErT). Moreover, we identified RAB22A as a direct target of miR-203 and RAB22A overexpression blocks the roles of miR-203 in osteosarcoma cell. Furthermore, we demonstrated that RAB22A expression was upregulated in human osteosarcoma cell lines and tissues. Take together, our results demonstrated that miR-203 act as a tumor suppressor miRNA through regulating RAB22A expression and suggested its involvement in osteosarcoma progression and carcinogenesis.     

A novel ER-localized transmembrane protein,EMC6, interacts with RAB5A and regulates cell autophagy

Autophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of the cytoplasm for delivery to the lysosome. Phosphatidylinositol 3-phosphate (PtdIns3P) produced by the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is essential for canonical autophagosome formation. RAB5A, a small GTPase localized to early endosomes, has been shown to associate with the class III PtdIns3K complex, regulate its activity and promote autophagosome formation. However, little is known about how endosome-localized RAB5A functions with the class III PtdIns3K complex. Here we identified a novel endoplasmic reticulum (ER)-localized transmembrane protein, ER membrane protein complex subunit 6 (EMC6), which interacted with both RAB5A and BECN1/Beclin 1 and colocalized with the omegasome marker ZFYVE1/DFCP1. It was shown to regulate autophagosome formation, and its deficiency caused the accumulation of autophagosomal precursor structures and impaired autophagy. Our study showed for the first time that EMC6 is a novel regulator involved in autophagy.     

Warburg Micro syndrome is caused by RAB18 deficiency or dysregulation

RAB18, RAB3GAP1, RAB3GAP2 and TBC1D20 are each mutated in Warburg Micro syndrome, a rare autosomal recessive multisystem disorder. RAB3GAP1 and RAB3GAP2 form a binary ‘RAB3GAP’ complex that functions as a guanine-nucleotide exchange factor (GEF) for RAB18, whereas TBC1D20 shows modest RAB18 GTPase-activating (GAP) activity in vitro. Here, we show that in the absence of functional RAB3GAP or TBC1D20, the level, localization and dynamics of cellular RAB18 is altered. In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated and less cytosolic than in control cells. These data suggest that RAB18 is a physiological substrate of TBC1D20 and contribute to a model in which a Rab-GAP can be essential for the activity of a target Rab. Together with previous reports, this indicates that Warburg Micro syndrome can be caused directly by loss of RAB18, or indirectly through loss of RAB18 regulators RAB3GAP or TBC1D20.     

乳腺浸润性微乳头状癌的研究现状

乳腺浸润性微乳头状癌(IMPC)是乳腺浸润性癌的一种特殊类型,其发生率低,临床表现及影像学特征与普通的乳腺浸润性导管癌没有显著区别。这种病理类型可与普通浸润性导管癌混合出现,也可表现为单纯的浸润性微乳头状癌。但浸润性微乳头状癌具有独特的组织学形态及分子结构,决定了其病理学分级较高、易于发生淋巴结转移的侵袭性生物学行为特点。多数浸润性微乳头状癌在影像学上表现为边缘不清的不规则肿块影,常伴有微小钙化。其特征性病理形态为细胞膜上皮抗原(EMA)在肿瘤细胞簇外周的细胞和基质中腔隙边缘特异性染色,同时显微镜下瘤细胞表面发现微绒毛结构,说明了瘤细胞簇周围的空隙样结构实际上是管状腔隙,瘤细胞呈"极向倒转"方式排列。IMPC具有高度淋巴血管浸润倾向,局部复发率高,是一种预后较差的类型。本文对近年来关于乳腺浸润性微乳头状癌的研究进展进行了综述。     

Regulation of RAB5C Is Important for the Growth Inhibitory Effects of MiR-509 in Human Precursor-B Acute Lymphoblastic Leukemia

MicroRNAs (miRs) regulate essentially all cellular processes, but few miRs are known to inhibit growth of precursor-B acute lymphoblastic leukemias (B-ALLs). We identified miR-509 via a human genome-wide gain-of-function screen for miRs that inhibit growth of the NALM6 human B-ALL cell line. MiR-509-mediated inhibition of NALM6 growth was confirmed by 3 independent assays. Enforced miR-509 expression inhibited 2 of 2 additional B-ALL cell lines tested, but not 3 non-B-ALL leukemia cell lines. MiR-509-transduced NALM6 cells had reduced numbers of actively proliferating cells and increased numbers of cells undergoing apoptosis. Using miR target prediction algorithms and a filtering strategy, RAB5C was predicted as a potentially relevant target of miR-509. Enforced miR-509 expression in NALM6 cells reduced RAB5C mRNA and protein levels, and RAB5C was demonstrated to be a direct target of miR-509. Knockdown of RAB5C in NALM6 cells recapitulated the growth inhibitory effects of miR-509. Co-expression of the RAB5C open reading frame without its 3′ untranslated region (3′UTR) blocked the growth-inhibitory effect mediated by miR-509. These findings establish RAB5C as a target of miR-509 and an important regulator of B-ALL cell growth with potential as a therapeutic target.     

RAB11A is essential for transport of the influenza virus genome to the plasma membrane

Influenza A virus assembly is a complex process that requires the intersection of pathways involved in transporting viral glycoproteins, the matrix protein, and viral genomes, incorporated in the viral ribonucleoprotein (vRNP) complex, to plasma membrane sites of virion formation. Among these virion components, the mechanism of vRNP delivery is the most incompletely understood. Here, we reveal a functional relationship between the cellular Rab11 GTPase isoform, RAB11A, and vRNPs and show that RAB11A is indispensable for proper vRNP transport to the plasma membrane. Using an immunofluorescence-based assay with a monoclonal antibody that recognizes nucleoprotein in the form of vRNP, we demonstrate association between RAB11A and vRNPs at all stages of vRNP cytoplasmic transport. Abrogation of RAB11A expression through small interfering RNA (siRNA) treatment or disruption of RAB11A function by overexpression of dominant negative or constitutively active proteins caused aberrant vRNP intracellular accumulation, retention in the perinuclear region, and lack of accumulation at the plasma membrane. Complex formation between RAB11A and vRNPs was further established biochemically. Our results uncover a critical host factor with an essential contribution to influenza virus genome delivery and reveal a potential role for RAB11A in the transport of ribonucleoprotein cargo.     

RAB31 marks and controls an ESCRT-independent exosome pathway

Exosomes are generated within the multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell membrane. The mechanisms of exosome biogenesis remain poorly explored. Here we identify that RAB31 marks and controls an ESCRT-independent exosome pathway. Active RAB31, phosphorylated by epidermal growth factor receptor (EGFR), engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form ILVs, which is independent of the ESCRT (endosomal sorting complex required for transport) machinery. Active RAB31 interacts with the SPFH domain and drives ILV formation via the Flotillin domain of flotillin proteins. Meanwhile, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing the fusion of MVEs with lysosomes and enabling the secretion of ILVs as exosomes. These findings establish that RAB31 has dual functions in the biogenesis of exosomes: driving ILVs formation and suppressing MVEs degradation, providing an exquisite framework to better understand exosome biogenesis.Subject terms: Small GTPases, Endosomes, Multivesicular bodies, Lysosomes, ESCRT     

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.     

一个与非小细胞肺癌转移相关的基因――RAB5A基因

采用mRNA差异展示技术（mRNA DD）研究具有相同细胞来源,但转移能力高低不同的人肺腺癌细胞系AGZY83-a（低转移）和Anip973(高转移),分析在两个细胞系中基因差异表达的情况,发现在高转移细胞系中有RAB5A基因的表达。该基因为蛋白质入胞信号的调控者,为RAS超家族成员。为进一步证实其转录表达的调控改变情况,以及RAB5A高表达的临床意义,进一步采用RT-PCR和免疫组织化学的方法检测了50例临床非小细胞肺癌的手术标本,结果表明,RAB5A的表达有随转移发生而增强的趋势,而RAB5A的蛋白表达程度在有转移的病例中明显增强(P<0.05)。 Abstract：　Using mRNA differential display (mRNA DD)techniques, we analyzed the differences of gene expression between two human lung adenocarcinoma cell lines,AGZY83-a and Anip973. Anip973 was isolated from AGZY83-a, but manifested much higher metastatic potential than the parent line. The results showed that there were significant differences on gene expression between the two cell lines and that there was over-expression of RAB5Agene in the Anip973 cell line. The product of RAB5Agene was recognized as signal regulators of endocytotic pathway and protein trafficking at the cell surface, and belong to a member of the RAS superfamily. Furthemore, we compared to the expression of RAB5Agene and RAB5Aprotein in clinical samples of 50 cases non-small lung carcinoma and nearby lymph node by means of RT-PCR and immunohistochemistry method. The results indicated that the high expression of RAB5Ain metastatic tumor and the enhancement level of RAB5Ain metastatic tumor and the enhancement level of expression were corresponded with the increase of metastatic degree. And there were significance of difference on the expression degree of RAB5Aprotein between non-small lung carcinoma with metastasis and non- metastasis (P<0.05).     

GANT61 plays antitumor effects by inducing oxidative stress through the miRNA-1286/RAB31 axis in osteosarcoma

Osteosarcoma (OS) is a rare malignancy of bone associated with poor clinical outcomes. The antitumor effects of GANT61 on OS is unclear. To investigate antitumor effects and mechanism of GANT61 in OS cells and xenograft model. Effects of GANT61 on cell viability, clone formation, cell cycle, apoptosis, migration, and invasion ability of OS cells were assessed. Reactive oxygen species (ROS) levels measured by dichlorofluorescein fluorescence were used to evaluate oxidative stress. The Xenograft model was constructed to investigate the antitumor effects of GANT61 in vivo. The microRNA (miRNA)-1286 was downregulated, while RAB31 upregulated in OS tissues and cells. GANT61 inhibited viability, migration, and invasion ability of OS cells (SaOS-2 and U2OS), and induced apoptosis and the ROS production, along with miRNA-1286 upregulation and RAB13 downregulation. After knockdown of miRNA-1286, GANT6-induced cell inhibition was attenuated, along with RAB31 upregulation. Inversely, miRNA-1286 overexpression downregulated RAB31. Dual-luciferase reporter assay verified that miR-1286 negatively targeted RAB13. Moreover, the knockdown of RAB31 stimulated apoptosis and ROS production while inhibited viability, migration, and invasion of GANT61-treated cells. In vivo experiments further confirmed that GANT61 inhibited tumor growth and RAB13 expression, but enhanced miRNA-1286. The study demonstrated that GANT61 inhibited cell aggressive phenotype and tumor growth by inducing oxidative stress through the miRNA-1286/RAB31 axis. Our findings provided a potential antitumor agent for the OS clinical treatment.     

Transforming growth factor beta (TGF-β) and inflammation in cancer

The transforming growth factor beta (TGF-β) has been studied with regard to the regulation of cell behavior for over three decades. A large body of research has been devoted to the regulation of epithelial cell and derivative carcinoma cell populations in vitro and in vivo. TGF-β has been shown to inhibit epithelial cell cycle progression and promote apoptosis that together significantly contribute to the tumor suppressive role for TGF-β during carcinoma initiation and progression. TGF-β is also able to promote an epithelial to mesenchymal transition that has been associated with increased tumor cell motility, invasion and metastasis. However, it has now been shown that loss of carcinoma cell responsiveness to TGF-β stimulation can also promote metastasis. Interestingly, enhanced metastasis in the absence of a carcinoma cell response to TGF-β stimulation has been shown to involve increased chemokine production resulting in recruitment of pro-metastatic myeloid derived suppressor cell (MDSC) populations to the tumor microenvironment at the leading invasive edge. When present, MDSCs enhance angiogenesis, promote immune tolerance and provide matrix degrading enzymes that promote tumor progression and metastasis. Further, the recruitment of MDSC populations in this context likely enhances the classic role for TGF-β in immune suppression since the MDSCs are an abundant source of TGF-β production. Importantly, it is now clear that carcinoma-immune cell cross-talk initiated by TGF-β signaling within the carcinoma cell is a significant determinant worth consideration when designing therapeutic strategies to manage tumor progression and metastasis.     

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2     

RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC.     

RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy

Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.     

Receptor-mediated internalization promotes increased endosome size and number in a RAB4- and RAB5-dependent manner

Despite their significance in receptor-mediated internalization and continued signal transduction in cells, early/sorting endosomes (EE/SE) remain incompletely characterized, with many outstanding questions that surround the dynamics of their size and number. While several studies have reported increases in EE/SE size and number resulting from endocytic events, few studies have addressed such dynamics in a methodological and quantitative manner. Herein we apply quantitative fluorescence microscopy to measure the size and number of EE/SE upon internalization of two different ligands: transferrin and epidermal growth factor. Additionally, we used siRNA knock-down to determine the involvement of 5 different endosomal RAB proteins (RAB4, RAB5, RAB8A, RAB10 and RAB11A) in EE/SE dynamics. Our study provides new information on the dynamics of endosomes during endocytosis, an important reference for researchers studying receptor-mediated internalization and endocytic events.