Expression Of Rab7 Research Articles

ZEB2 stably represses RAB25 expression through epigenetic regulation by SIRT1 and DNMTs during epithelial-to-mesenchymal transition

BackgroundEpithelial mesenchymal transition (EMT) is tightly regulated by a network of transcription factors (EMT-TFs). Among them is the nuclear factor ZEB2, a member of the zinc-finger E-box binding homeobox family. ZEB2 nuclear localization has been identified in several cancer types, and its overexpression is correlated with the malignant progression. ZEB2 transcriptionally represses epithelial genes, such as E-cadherin (CDH1), by directly binding to the promoter of the genes it regulates and activating mesenchymal genes by a mechanism in which there is no full agreement. Recent studies showed that EMT-TFs interact with epigenetic regulatory enzymes that alter the epigenome, thereby providing another level of control. The role of epigenetic regulation on ZEB2 function is not well understood. In this study, we aimed to characterize the epigenetic effect of ZEB2 repressive function on the regulation of a small Rab GTPase RAB25.ResultsUsing cellular models with conditional ZEB2 expression, we show a clear transcriptional repression of RAB25 and CDH1. RAB25 contributes to the partial suppression of ZEB2-mediated cell migration. Furthermore, a highly significant reverse correlation between RAB25 and ZEB2 expression in several human cancer types could be identified. Mechanistically, ZEB2 binds specifically to E-box sequences on the RAB25 promoter. ZEB2 binding is associated with the local increase in DNA methylation requiring DNA methyltransferases as well as histone deacetylation (H3K9Ac) depending on the activity of SIRT1. Surprisingly, SIRT1 and DNMTs did not interact directly with ZEB2, and while SIRT1 inhibition decreased the stability of long-term repression, it did not prevent down-regulation of RAB25 and CDH1 by ZEB2.ConclusionsZEB2 expression is resulting in drastic changes at the chromatin level with both clear DNA hypermethylation and histone modifications. Here, we revealed that SIRT1-mediated H3K9 deacetylation helps to maintain gene repression but is not required for the direct ZEB2 repressive function. Targeting epigenetic enzymes to prevent EMT is an appealing approach to limit cancer dissemination, but inhibiting SIRT1 activity alone might have limited effect and will require drug combination to efficiently prevent EMT.

Quercetin ameliorates autophagy in alcohol liver disease associated with lysosome through mTOR-TFEB pathway

This study aimed to investigate the effects of quercetin that is natural functional component in food on lysosome damage-mediated autophagy dysfunction in ALD and its possible underlying mechanisms. The C57BL/6J mice were divided into four groups and pair-fed with either regular or ethanol-containing Lieber De Carli liquids diets for 15 weeks. Quercetin was received by gavage. According to the purpose of experiments, primary hepatocytes were pretreated with various pharmacological reagents. Results showed that quercetin alleviated chronic ethanol consumption induced liver injury and autophagic flux suppression. Quercetin decreased the abnormal LC3-II and p62 accumulation and increased the expression of LAMP1, LAMP2 and Rab7. Besides, quercetin reversed the inhibition of TFEB nuclear translocation incited by ethanol and exhibited similar effect to Torin 1 (mTOR activity inhibitor) which could promote TFEB nuclear translocation. Thus, regulating mTOR-TFEB pathway may be a major mechanism of quercetin for ameliorating lysosomal autophagy dysfunction induced by ethanol.

MiR-384 suppressed renal cell carcinoma cell proliferation and migration through targeting RAB23.

microRNAs (miRNAs) are noncoding, short, and endogenous RNAs that play crucial roles in tumor progression at the post-transcriptional level. Here, we studied the role of miR-384 in the pathogenesis of renal cell carcinoma (RCC). We demonstrated that miR-384 expression was downregulated in the RCC specimens compared with nontumor specimens. Moreover, we showed that RAB23 expression was upregulated in the RCC tissues compared with nontumor tissues. Furthermore, we demonstrated that low expression of miR-384 was correlated with high levels of RAB23 in RCC tissues. We also demonstrated that the RAB23 was a direct target gene of miR-384 in RCC cells. In addition, overexpression of miR-384 suppressedRCC cell proliferation, cell cycle, and cell migration. Furthermore, ectopic expression of RAB23 promotedRCC cell proliferation, cell cycle, and cell migration. These data suggested that miR-384 played a tumor suppressor microRNA in the development of RCC partly through inhibiting RAB23 expression.

Fate of the Fc fusion protein aflibercept in retinal endothelial cells: competition of recycling and degradation

PurposeIntravitreal injection of the VEGF-binding protein aflibercept is widely used to treat various ocular diseases. In vitro, immortalized bovine retinal endothelial cells (iBREC) take up and transport aflibercept through the cell layer in a serum-dependent manner, likely mediated through the neonatal Fc receptor (FcRn), but degradation of the Fc domain-containing protein might be a competing intracellular process. Therefore, aflibercept’s associations with proteins either involved in FcRn-mediated transport or in the lysosomal pathway were studied.MethodsConfluent iBREC pre-cultivated with or without FBS were exposed for 4 h to in vivo achievable 250 μg/ml aflibercept, before cells were harvested for immunofluorescence staining or preparation of protein extracts. Intracellular localization of aflibercept and putative co-localizations with proteins involved in transport of IgG/FcRn complexes, i.e., endosomal Rab4 and Rab11, components of the cytoskeleton, motor proteins, or with marker proteins characteristic of multivesicular bodies or lysosomes were assessed by co-immunofluorescence stainings. Amounts of expressed endogenous proteins and of internalized aflibercept were determined by Western blot analyses.ResultsAflibercept-specific perinuclear staining overlapped with that of the motor protein dynein whereas double staining with an anti-kinesin antibody resulted in a patchy pattern. In addition, aflibercept was typically present close to microtubules and often co-localized with α-tubulin. Rab4 and Rab11 stainings partly overlapped with the perinuclear staining of aflibercept whereas co-localization with Rab7 (in late endosomes/lysosomes) was only rarely seen. Interestingly, aflibercept but not the IgG bevacizumab broadly co-localized with the cation-independent mannose 6-phosphate receptor characteristic of multivesicular endosomes. In accordance with partial degradation beside transcytosis, the amount of intracellular aflibercept increased when cells were treated with protease inhibitors MG-132 or MG-101. Serum-deprived iBREC expressed less Rab11 and dynein but slightly more Rab4.ConclusionAfter uptake by iBREC, aflibercept is present in organelles associated with FcRn-mediated transport, but part of the protein is subject to degradation. Transport inhibition of aflibercept during cultivation without FBS is likely a consequence of an attenuated exocytosis due to decreased expression of Rab11.

EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation

ABSTRACTEBV has been reported to impair monocyte in vitro differentiation into dendritic cells (DCs) and reduce cell survival. In this study, we added another layer of knowledge to this topic and showed that these effects correlated with macroautophagy/autophagy, ROS and mitochondrial biogenesis reduction. Of note, autophagy and ROS, although strongly interconnected, have been separately reported to be induced by CSF2/GM-CSF (colony stimulating factor 2) and required for CSF2-IL4-driven monocyte in vitro differentiation into DCs. We show that EBV infects monocytes and initiates a feedback loop in which, by inhibiting autophagy, reduces ROS and through ROS reduction negatively influences autophagy. Mechanistically, autophagy reduction correlated with the downregulation of RAB7 and ATG5 expression and STAT3 activation, leading to the accumulation of SQSTM1/p62. The latter activated the SQSTM1-KEAP1- NFE2L2 axis and upregulated the anti-oxidant response, reducing ROS and further inhibiting autophagy. ROS decrease correlated also with the reduction of mitochondria, the main source of intracellular ROS, achieved by the downregulation of NRF1 and TFAM, mitochondrial biogenesis transcription factors. Interestingly, mitochondria supply membranes and ATP required for autophagy execution, thus their reduction may further reduce autophagy in EBV-infected monocytes. In conclusion, this study shows for the first time that the interconnected reduction of autophagy, intracellular ROS and mitochondria mediated by EBV switches monocyte differentiation into apoptosis, giving new insights into the mechanisms through which this virus reduces immune surveillance.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CSF2: colony stimulating factor 2; CT: control; CYCS (cytochrome C: somatic); DCs: dendritic cells; EBV: Epstein-Barr virus; GSR: glutathione-disulfide reductase; KEAP1: kelch like ECH associated protein 1; IL4: interleukin 4; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MET: metformin; NAC: N-acetylcysteine; NFE2L2/NRF2 nuclear factor: erythroid 2 like 2; NRF1 (nuclear respiratory factor 1); clPARP1: cleaved poly(ADP-ribose) polymerase; Rapa: Rapamycin; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TFAM: (transcription factor A: mitochondrial); TUBA1A: tubulin alpha 1a.

Inhibitory effect of microRNA-455-5p on biological functions of esophageal squamous cell carcinoma Eca109 cells via Rab31.

The aim of the present study was to examine microRNA (miRNA or miR)-455-5p expression in esophageal squamous cell carcinoma (ESCC) at the tissue and cellular levels in order to elucidate its biological roles. A total of 60 patients with ESCC were enrolled in the present study and reverse transcription-quantitative polymerase chain reaction was used to measure the expression of miR-455-5p. ESCC Eca109 cells were transfected with miR-NC, miR-455-5p mimics or inhibitor and a Cell Counting Kit-8 assay was used to assess proliferation. To investigate the migration and invasion abilities of Eca109 cells, Transwell and Matrigel assays were performed. Western blotting was employed to measure Rab31 protein expression, while a rescue assay was utilized to study the biological roles of miR-455-5p and Rab31 in Eca109 cells. To determine whether Rab31 is a direct target of miR-455-5p, a dual luciferase reporter assay was performed. The results revealed that miR-455-5p expression was decreased in ESCC tissues and was negatively correlated with metastasis and pathogenesis. In vitro overexpression of miR-455-5p inhibited the proliferation, migration and invasion of ESCC Eca109 cells. Furthermore, miR-455-5p regulated the expression of Rab31 protein in Eca109 cells. Rab31 overexpression promoted the proliferation, migration and invasion of Eca109 cells. Luciferase reporter assay results revealed that miR-455-5p is able to bind with the 3′-untranslated region of Rab31 mRNA to regulate its expression. In summary, the results of the present study suggest that miR-455-5p expression is decreased in ESCC tissues and is miR-455-5p is negatively correlated with lymphatic metastasis and differentiation. As a tumor-suppressor gene, miR-455-5p inhibits the proliferation, migration and invasion of ESCC Eca109 cells by suppressing the expression of Rab31.

Rab14 overexpression regulates gemcitabine sensitivity through regulation of Bcl-2 and mitochondrial function in pancreatic cancer.

Rab family protein Rab14 has been implicated in the development of human cancers. To date, its expression pattern, biological function, and potential mechanism in pancreatic cancer have not been explored. In this study, we analyzed Rab14 expression in 103 cases of pancreatic cancer tissues using immunohistochemistry (IHC) and found that Rab14 was overexpressed in 41/103 cases (39.8%). Rab14 overexpression correlated with the advanced stage. Moreover, elevated Rab14 levels indicated poor prognosis of patients with pancreatic cancers. We used BxPC-3 and Capan-2 respectively for plasmid and siRNA transfection. MTT and colony formation assays showed that Rab14 transfection increased cell proliferation and colony formation in BxPC-3 cells. Rab14 siRNA knockdown inhibits proliferation and colony formation ability in Capan-2 cell line. Cell cycle analysis showed that Rab14 facilitated cell cycle progression. Matrigel invasion assay showed that Rab14 promoted BxPC-3 cell invasion while its depletion inhibited Capan-2 cell invasion. In addition, MTT and AnnexinV/PI analysis demonstrated that overexpression of Rab14 reduced gemcitabine sensitivity which conversely was increased by Rab14 knockdown. We also demonstrated that Rab14 upregulated mitochondrial membrane potential (MMP) while its depletion downregulated MMP during gemcitabine treatment. In addition, western blotting revealed that Rab14 overexpression upregulated cyclin D1, cyclin A, cyclin E, p-Rb, and Bcl-2 and downregulated p21. Rab14 also downregulated caspase3, PARP cleavage, and cytochrome c release. In conclusion, our data indicated that Rab14 was overexpressed in pancreatic cancer and promotes growth and gemcitabine resistance, possibly through regulation of mitochondrial function and Bcl-2.

Integrative functional genomics identifies regulatory genetic variant modulating RAB31 expression and altering susceptibility to breast cancer.

Despite the successes of genome-wide association study (GWAS) in identifying breast cancer (BC) risk-associated variants, only a small fraction of the heritability can be explained. The greatest challenge in the post-GWAS is to identify causal variants and underlying mechanisms responsible for BC susceptibility. In this study, we integrated functional genomic data from ENCODE ChIP-seq, ANNOVAR, and the TRANSFAC matrix to identify potentially regulatory variants with modulating FOXA1-binding affinity across the whole genome, and then conducted a two-stage case-control study including 2164 cases and 2382 controls to investigate the associations between candidate SNPs and BC susceptibility. We identified a BC susceptibility SNP, rs6506689 G>T, with an odds ratio (OR) of 1.23 (95% confidence interval = 1.07-1.40, P = 0.003) under a dominant model in the combined study. Biological assays indicated that the germline G>T variation at rs6506689 creates a FOXA1-binding site and up-regulates the expression of RAB31, thus playing an important role in the development of BC. Our results highlight the importance of regulatory genetic variants in the development of BC by influencing TF-DNA interaction and provide critical insights to pinpoint causal genetic variants.

Innate Immune Signals Induce Anterograde Endosome Transport Promoting MHC Class I Cross-Presentation

Both cross-presentation of antigens by dendritic cells, a key pathway triggering Tcell immunity and immune tolerance, and survival of several pathogens residing in intracellular vacuoles are intimately linked to delayed maturation of vesicles containing internalized antigens and microbes. However, how early endosome or phagosome identity is maintained is incompletely understood. We show that Toll-like receptor 4 (TLR4) and Fc receptor ligation induces interaction of the GTPase Rab14 with the kinesin KIF16b mediating plus-end-directed microtubule transport of endosomes. As a result, Rab14 recruitment to phagosomes delays their maturation and killing of an internalized pathogen. Enhancing anterograde transport by overexpressing Rab14, promoting the GTP-bound Rab14 state, or inhibiting retrograde transport upregulates cross-presentation. Conversely, reducing Rab14 expression, destabilizing Rab14 endosomes, and inhibiting anterograde microtubule transport by Kif16b knockdown compromise cross-presentation. Therefore, regulation of early endosome trafficking by innate immune signals is a critical parameter in cross-presentation by dendritic cells.

Rab GTPase regulation of bacteria and protozoa phagocytosis occurs through the modulation of phagocytic receptor surface expression

Phagocytosis of invading microorganisms by professional phagocytic cells has a central role in innate immunity. However, several microorganisms developed strategies to subvert this process. Previously, we reported that bacteria and protozoa modulate differently the expression of Rab GTPases. Moreover, our results suggested that this modulation can contribute to avoid phagocytosis. Here, we investigated the mechanism by which the malaria parasite Plasmodium berghei and the bacterium Escherichia coli subvert phagocytosis through the modulation of Rab14 or Rab9a expression, respectively. We first confirmed that the scavenger receptor CD36 and the Toll-like receptor (TLR) 4 are required for the phagocytosis of P. berghei and E. coli, respectively. Interestingly, we observed that Rab14 silencing leads to an increase in the surface expression of CD36 in macrophages, which can explain the increase in the phagocytosis of P. berghei we reported previously. Similar results were obtained for Rab9a and TLR4, i.e. Rab9a silencing causes an upregulation of TLR4 surface expression in macrophages. Furthermore, we found that the decrease in the internalization of CD36 and TLR4, upon Rab14 or Rab9a silencing, respectively, can explain the increase in the surface levels of these receptors. Thus, our studies provide evidence that the modulation of phagocytosis caused by changes in Rab expression is operated, at least partly through changes in the surface levels of phagocytic receptors.

Nicastrin/miR-30a-3p/RAB31 Axis Regulates Keratinocyte Differentiation by Impairing EGFR Signaling in Familial Acne Inversa

Nicastrin (NCSTN) mutations are associated with familial acne inversa (AI), and emerging evidence suggests that microRNAs (miRNAs) are involved in various skin diseases. However, whether NCSTN mutations affect miRNA levels and their subsequent signaling pathways in familial AI patients has not been studied. We aimed to elucidate the relationship between NCSTN mutations and familial AI pathogenesis by investigating differential miRNA expression and their related pathways. Combined with miRNA microarray data from familial AI patients, Ncstn keratinocyte-specific-knockout (NcstnΔKC) mice and bioinformatics predictions showed that NCSTN mutations led to decreased miR-30a-3p levels, which negatively regulated RAB31 expression. Moreover, enhanced RAB31 levels accelerated degradation of activated EGFR, leading to abnormal differentiation in keratinocytes. The impaired EGFR signaling and its effects on epidermal differentiation were also observed in familial AI patients and NcstnΔKC mice. Thus, our study showed that miR-30a-3p/RAB31/EGFR signaling pathway may play a key role in the pathogenesis of familial AI with NCSTN mutations.

Rab11 Functions as an Oncoprotein via Nuclear Factor kappa B (NF-κB) Signaling Pathway in Human Bladder Carcinoma.

BackgroundElevated expression of Rab11 has been reported in different human cancers, including human bladder carcinoma. This study, we investigated the biological effects and mechanism of Rab11 overexpression in human bladder carcinoma for the first time.Material/MethodsRab11 expression in bladder cancer tissues was detected using immunohistochemistry and Western blot analysis. Then, Rab11 expression was inhibited in T24 cells and it was overexpressed in BIU-87 cells. The effects of Rab11 perturbations on cell growth rate and invasion were analyzed by CCK8, cell cycle assay, and matrix gel invasion assay. MMP-9, cyclin E, and cyclin D1 levels were studied using Western blot and qPCR. NF-κB activity was studied by luciferase assay.ResultsHigh expression of Rab11 was detected in 41.5% (66/159) of tumor specimens. We found a significant correlation between high Rab11 expression and depth of tumor invasion (P=0.004). Rab11 overexpression was observed to promote the growth rate and invasiveness of cancer cells through upregulation of MMP9, cyclin E, and cyclin D1 levels. Rab11 overexpression further elevated NF-κB reporter activity and enhanced p-IκB expression. Use of BAY 11-7082, a noted NF-κB inhibitor, partially abolished overexpression of MMP9 and cyclin D1 by Rab11.ConclusionsOur research proved that high Rab11 expression enhances cellular multiplication and invasiveness of bladder cancer, possibly by regulating the NF-κB signaling pathway.

Decreased expression of miR‑490‑3p in colorectal cancer predicts poor prognosis and promotes cell proliferation and invasion by targeting RAB14.

Growing evidence indicates a potential role for miR‑490‑3p in tumorigenesis. However, its function in colorectal carcinoma (CRC) remains undefined. In this study, miR‑490‑3p was markedly downregulated in fifty colorectal cancer tissue samples compared with the corresponding adjacent non‑cancerous specimens, by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of miR‑490‑3p were closely associated with tumor differentiation and distant metastasis. In addition, both Kaplan-Meier and multivariate analyses indicated CRC patients with elevated miR‑490‑3p amounts had prolonged overall survival. Overexpression of miR‑490‑3p markedly reduced proliferation, colony formation and invasion in CRC cells by enhancing apoptosis and promoting G2/M phase arrest. Furthermore, ectopic expression of miR‑490‑3p resulted in decreased expression of RAB14, which was directly targeted by miR‑490‑3p, as shown by the dual-luciferase reporter gene assay. Finally, in a nude mouse model, miR‑490‑3p overexpression significantly suppressed the growth of CRC cells. The above results indicated that miR‑490‑3p might constitute a prognostic indicator and a novel molecular target for miRNA-based CRC therapy.

HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System.

The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

MicroRNA-16 suppressed the invasion and migration of osteosarcoma by directly inhibiting RAB23.

Osteosarcoma is the most frequent primary bone malignancy that affects young adults and adolescents around the world. Increasing evidence suggests that dysfunctions of microRNAs (miRNAs) used to play an important role in human cancers. We aimed at evaluating the potential function of miR-16 and verify its influence on the function of RAB23 in osteosarcoma. miR-16 expressions in osteosarcoma tissues and cell lines were examined using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Transwell chambers were conducted to detect the miR-16 effects on osteosarcoma cells migration and invasion. Meanwhile, Western blot and luciferase assays were performed to validate RAB23 as miR-16 targets. miR-16 was down-regulated in osteosarcoma cell lines (MG63, SAOS-2, U2OS, and SOSP-9607) and osteosarcoma specimens, while RAB23 expression was higher in tumor tissues. Ectopic over-expression of miR-216 in osteosarcoma cells could inhibit cells migration and invasion. RAB23 was confirmed as a direct target of miR-16 and the inverse relationship between them was also observed. Over-expression of RAB23 ablates the inhibitory effects of miR-16. miR-16 inhibited cancer migration and invasion, and promoted the RAB23 expression in osteosarcoma. This newly identified miR-16/RAB23 axis may provide new insight into the pathogenesis and represents a potential therapeutic target for osteosarcoma.

The breast cancer antigen 5T4 interacts with Rab11, and is a target and regulator of Rab11 mediated trafficking.

5T4 is a transmembrane glycoprotein with limited expression in normal adult tissues and expression in some solid tumours. It is unclear whether 5T4 is preferentially expressed by stem or differentiated cell types. Modes of 5T4 regulation are unknown despite its ongoing development as a cancer immunotherapy target. Our aims were to clarify the differentiation status of 5T4 expressing cells in breast cancer and to understand the mechanism underlying 5T4 membrane presentation. We analysed 5T4 expression in breast cancer cell populations by flow cytometery and found that 5T4 is highly expressed on differentiated cells, where it localizes to focal adhesions. Using immunoprecipitation and mass spectrometry, we identified interactions between 5T4 and the membrane trafficking proteins Rab11, Rab18 and ARF6. Mechanistically we found that Rab11 and Rab18 have oppositional roles in controlling expression and surface presentation of 5T4. 5T4 depletion stabilizes Rab11 protein expression with a consequent stimulation transferrin surface labelling, indicating that 5T4 represses endocytic activity. Successful immunotherapeutic targeting of 5T4 requires surface presentation and different immunotherapy strategies require surface presentation versus endocytosis. While breast cancer cells with high 5T4 surface expression and rapid cell surface turnover would be susceptible to antibody-drug conjugates that rely on intracellular release, 5T4 positive cells with lower expression or lower turnover may still be responsive to T-cell mediated approaches. We find that endocytosis of 5T4 is strongly Rab11 dependent and as such Rab11 activity could affect the success or failure of 5T4-targetted immunotherapy, particularly for antibody-drug conjugate approaches. In fact, 5T4 itself represses Rab11 expression. This newly uncovered relationship between Rab11 and 5T4 suggests that breast tumours with high 5T4 expression may not have efficient endocytic uptake of 5T4-targetted immunotherapeutics. This should be considered when selecting amongst the different types of immunotherapies.

MiR‐577 suppresses epithelial‐mesenchymal transition and metastasis of breast cancer by targeting Rab25

BackgroundMicroRNAs can act as both tumor suppressor genes and oncogenes and participate in cell proliferation, metastasis, and apoptosis. Low levels of miR‐577 are found in several cancers, for example, thyroid carcinoma, glioblastoma, and hepatocellular carcinoma. The aim of this study was to investigate the effect of miR‐577 on breast cancer (BC).MethodsThe relative level of miR‐577 in 120 BC tissues and cells was detected by real‐time PCR. MDA‐MB‐231 cells with upregulated miR‐577 and MCF‐7 cells with downregulated miR‐577 were established. Transwell invasion assays were used to examine the invasiveness of cells. Epithelial‐mesenchymal transition (EMT) markers were evaluated by immunofluorescence and Western blot. Targeted combinations of miR‐577 and Rab25 were analyzed by luciferase assays. Xenograft models were used to examine the effect of miR‐577 on BC metastasis.Results MiR‐577 expression was significantly suppressed in BC tissues. Tumor size, tumor stage, and lymphatic metastasis were attributed to miR‐577 expression. Moreover, miR‐577 overexpression strongly inhibited the invasiveness and EMT of BC cells in vitro. MiR‐577 directly regulated Rab25 in BC. Rab25 upregulation by miR‐577 decreased the levels of E‐cadherin and increased the levels of Vimentin. Notably, Rab25 knockdown inhibited BC invasion; however, an increase in Rab25 counteracted the invasive effect of miR‐577 in BC.ConclusionResults indicated that miR‐577 suppressed EMT by inhibiting Rab25 expression in BC. MiR‐577 and Rab25 are considered potential targets of BC treatment.

Abstract P6-05-02: Targeting vesicular trafficking machinery for breast cancer therapeutics

Abstract The Rab GTPase family is a key regulator of vesicular import, transport and export. Deregulation of vesicular trafficking promotes undesirable intercellular cross talk via exosomes that contribute to cancer pathophysiology. Preclinical and clinical studies underscore a role for exosomes and their cargo in breast cancer development, progression, metastasis, and resistance to therapy. While many studies have approached the role of exosomes in cancer pathophysiology, the 'rules' governing their production and cargo sorting represents a major unexplored gap in knowledge. Exosomes arise from internal budding of specific endosomal compartments. Rab11 GTPase family members comprised of Rab11a, Rab11b and Rab25 are critical regulators of endocytic cargo trafficking specifically of apical and recycling endosomes. Previously, our lab has shown that Rab25 is amplified and contributes to oncogenesis in both ovarian and Luminal B breast cancers. However, Rab25 is lost and when re-expressed ectopically, it inhibits invasion and migration in Claudin Low tumors suggesting a cell-lineage and context dependent function. Importantly, while in Luminal B cancer, Rab25 and its downstream effector RCP (Rab coupling protein), collaborate in driving oncogenesis, in Her2+ cancers, RCP expression is protective. Further our group recently uncovered the presence of a stroma enriched “reactive protein signature” in ER+/Her- group that is indicative of good outcome for patients. Indeed, many of the “reactive signature” proteins are reported in exosomal cargo by others. Taking together, our ongoing study utilizes a panel of breast cancer cell lines representing ER+ve/Her2+; ER+/Her-, ER-/Her2+, and ER-/Her- to evaluate the role of Rab11 family in modulating vesicular trafficking that alters the exosome biology and how that may contribute to tumor- stroma communications. Our preliminary results show that indeed over expression of Rab25 differentially alters size and number of extracellular vesicles in Luminal B cancers when compared to the Claudin Low subtype. Using a novel class of peptides that interrupt the cellular functions 25, we are investigating if Rab25 expression can be associated with an exosomal cargo profile in each subtype of breast cancer, which could then serve as a biomarker for tumor-stroma interaction. Citation Format: Mitra S, Vykoukal J, Jeong K, Mills G. Targeting vesicular trafficking machinery for breast cancer therapeutics [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-05-02.

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.