Adaptive stimulation of macropinocytosis overcomes aspartate limitation in cancer cells under hypoxia

ABSTRACTStress-adaptive mechanisms enable tumor cells to overcome metabolic constraints in nutrient and oxygen poor tumors. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumor growth are poorly understood. Here, using a combination of metabolomics and CRISPR-based genetic screens, we identify GOT2-catalyzed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumor cells under hypoxic culture conditions. In contrast, GOT2-catalyzed aspartate synthesis is dispensable for pancreatic tumor formation in vivo. The dependence of pancreatic tumor cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRas-dependent, and is mediated by hypoxia inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9) through the cooption of the bicarbonate-macropinocytosis signaling axis. Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRas tumors can overcome nutrient deprivation under hypoxic conditions.

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Pancreatic tumor cell metastasis is restricted by MT1-MMP binding protein MTCBP-1

The Journal of Cell Biology ◽

10.1083/jcb.201802032 ◽

2018 ◽

Vol 218 (1) ◽

pp. 317-332 ◽

Cited By ~ 10

Author(s):

Li Qiang ◽

Hong Cao ◽

Jing Chen ◽

Shaun G. Weller ◽

Eugene W. Krueger ◽

...

Keyword(s):

Tumor Cells ◽

Pancreatic Tumor ◽

Binding Protein ◽

Critical Role ◽

Inverse Correlation ◽

Pancreatic Tumors ◽

Matrix Remodeling ◽

Peripheral Tissues

The process by which tumor cells mechanically invade through surrounding stroma into peripheral tissues is an essential component of metastatic dissemination. The directed recruitment of the metalloproteinase MT1-MMP to invadopodia plays a critical role in this invasive process. Here, we provide mechanistic insight into MT1-MMP cytoplasmic tail binding protein 1 (MTCBP-1) with respect to invadopodia formation, matrix remodeling, and invasion by pancreatic tumor cells. MTCBP-1 localizes to invadopodia and interacts with MT1-MMP. We find that this interaction displaces MT1-MMP from invadopodia, thereby attenuating their number and function and reducing the capacity of tumor cells to degrade matrix. Further, we observe an inverse correlation between MTCBP-1 and MT1-MMP expression both in cultured cell lines and human pancreatic tumors. Consistently, MTCBP-1–expressing cells show decreased ability to invade in vitro and metastasize in vivo. These findings implicate MTCBP-1 as an inhibitor of the metastatic process.

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Hypoxia Preconditioning of Human MSCs: a Direct Evidence of HIF-1α and Collagen Type XV Correlation

Cellular Physiology and Biochemistry ◽

10.1159/000495869 ◽

2018 ◽

Vol 51 (5) ◽

pp. 2237-2249 ◽

Cited By ~ 5

Author(s):

Elisabetta Lambertini ◽

Letizia Penolazzi ◽

Marco Angelozzi ◽

Leticia Scussel Bergamin ◽

Cristina Manferdini ◽

...

Keyword(s):

Collagen Type ◽

Therapeutic Potential ◽

Hypoxia Inducible Factor ◽

Culture Conditions ◽

Luciferase Reporter ◽

Osteogenic Potential ◽

Human Mscs ◽

Short Period ◽

Hypoxia Preconditioning

Background/Aims: Mesenchymal stromal cells (MSCs) hold considerable promise in bone tissue engineering, but their poor survival and potency when in vivo implanted limits their therapeutic potential. For this reason, the study on culture conditions and cellular signals that can influence the potential therapeutic outcomes of MSCs have received considerable attention in recent years. Cell maintenance under hypoxic conditions, in particular for a short period, is beneficial for MSCs, as low O2 tension is similar to that present in the physiologic niche, however the precise mechanism through which hypoxia preconditioning affects these cells remains unclear. Methods: In order to explore what happens during the first 48 h of hypoxia preconditioning in human MSCs (hMSCs) from bone marrow, the cells were exposed to 1.5% O2 tension in the X3 Hypoxia Hood and Culture Combo – Xvivo System device. The expression modulation of critical genes which could be good markers of increased osteopotency has been investigated by Western blot, immunufluorescence and ELISA. Luciferase reporter assay and Chromatin immunoprecipitation was used to investigate the regulation of the expression of Collagen type XV (ColXV) gene. Results: We identified ColXV as a new low O2 tension sensitive gene, and provided a novel mechanistic evidence that directly HIF-1α (hypoxia-inducible factor-1 alpha) mediates ColXV expression in response to hypoxia, since it was found specifically in vivo recruited at ColXV promoter, in hypoxia-preconditioned hMSCs. This finding, together the evidence that also Runx2, VEGF and FGF-2 expression increased in hypoxia preconditioned hMSCs, is consistent with the possibility that increased ColXV expression in response to hypoxia is mediated by an early network that supports the osteogenic potential of the cells. Conclusion: These results add useful information to understand the role of a still little investigated collagen such as ColXV, and identify ColXV as a marker of successful hypoxia preconditioning. As a whole, our data give further evidence that hypoxia preconditioned hMSCs have greater osteopotency than normal hMSCs, and that the effects of hypoxic regulation of hMSCs activities should be considered before they are clinically applied.

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Targeting galectin-1 inhibits pancreatic cancer progression by modulating tumor–stroma crosstalk

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1722434115 ◽

2018 ◽

Vol 115 (16) ◽

pp. E3769-E3778 ◽

Cited By ~ 37

Author(s):

Carlos A. Orozco ◽

Neus Martinez-Bosch ◽

Pedro E. Guerrero ◽

Judith Vinaixa ◽

Tomás Dalotto-Moreno ◽

...

Keyword(s):

Pancreatic Cancer ◽

Tumor Cells ◽

Cancer Progression ◽

Pancreatic Tumor ◽

Therapeutic Potential ◽

Tumor Formation ◽

Pancreatic Stellate Cells ◽

Ductal Adenocarcinoma ◽

Migration And Invasion ◽

Regulatory Pathways

Pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal tumor types, with extremely low survival rates due to late diagnosis and resistance to standard therapies. A more comprehensive understanding of the complexity of PDA pathobiology, and especially of the role of the tumor microenvironment in disease progression, should pave the way for therapies to improve patient response rates. In this study, we identify galectin-1 (Gal1), a glycan-binding protein that is highly overexpressed in PDA stroma, as a major driver of pancreatic cancer progression. Genetic deletion of Gal1 in a Kras-driven mouse model of PDA (Ela-KrasG12Vp53−/−) results in a significant increase in survival through mechanisms involving decreased stroma activation, attenuated vascularization, and enhanced T cell infiltration leading to diminished metastasis rates. In a human setting, human pancreatic stellate cells (HPSCs) promote cancer proliferation, migration, and invasion via Gal1-driven pathways. Moreover, in vivo orthotopic coinjection of pancreatic tumor cells with Gal1-depleted HPSCs leads to impaired tumor formation and metastasis in mice. Gene-expression analyses of pancreatic tumor cells exposed to Gal1 reveal modulation of multiple regulatory pathways involved in tumor progression. Thus, Gal1 hierarchically regulates different events implicated in PDA biology including tumor cell proliferation, invasion, angiogenesis, inflammation, and metastasis, highlighting the broad therapeutic potential of Gal1-specific inhibitors, either alone or in combination with other therapeutic modalities.

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Induction of clonal monocyte-macrophage tumors in vivo by a mouse c-myc retrovirus: rearrangement of the CSF-1 gene as a secondary transforming event.

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.664 ◽

1987 ◽

Vol 7 (2) ◽

pp. 664-671 ◽

Cited By ~ 27

Author(s):

W R Baumbach ◽

E R Stanley ◽

M D Cole

Keyword(s):

Tumor Cells ◽

Gene Rearrangement ◽

Tumor Formation ◽

Dna Rearrangement ◽

Phagocytic Cells ◽

Proviral Integration ◽

Myc Oncogene ◽

Myc Gene ◽

Stimulating Factor

A mouse retrovirus containing the c-myc oncogene was found to induce tumors of mononuclear phagocytic cells in vivo. All tumors expressed the c-myc retroviral gene but not the endogenous c-myc gene (with one exception), and virtually all tumors were clonal with a unique proviral integration. This observation, coupled with a lag time in tumor formation, suggests that a second event, in addition to c-myc proviral integration, is necessary for the generation of neoplastic cells in vivo. All of the tumor cells expressed high levels of mRNA for both the putative colony-stimulating factor 1 (CSF-1) receptor (c-fms proto-oncogene product), as well as the c-fos proto-oncogene. Although all of the tumor cells proliferated in culture without the addition of exogenous CSF-1, which is required for the proliferation of primary macrophages partially transformed by the same c-myc retrovirus, several phenotypes were observed with respect to the expression of CSF-1 and granulocyte-macrophage CSF and to their growth factor responsiveness. The proliferation of one tumor, which secreted high levels of CSF-1, was blocked by specific anti-CSF-1 serum. This tumor was found to express altered CSF-1 mRNA and to have a DNA rearrangement at the CSF-1 locus. In this particular case, the data indicate that a CSF-1 gene rearrangement was the secondary event in development of the tumor. The pleiotropy of phenotypes among the other tumors indicated that there are a variety of other mechanisms for such secondary events which can be investigated with this system.

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Activating transcription factor 2 increases transactivation and protein stability of hypoxia-inducible factor 1α in hepatocytes

Biochemical Journal ◽

10.1042/bj20090371 ◽

2009 ◽

Vol 424 (2) ◽

pp. 285-296 ◽

Cited By ~ 16

Author(s):

Jeong Hae Choi ◽

Hyun Kook Cho ◽

Yung Hyun Choi ◽

JaeHun Cheong

Keyword(s):

Transcription Factor ◽

Protein Stability ◽

Gene Transcription ◽

Hypoxia Inducible Factor ◽

Protein Stabilization ◽

Hypoxic Conditions ◽

Tumour Suppressor Protein ◽

Activating Transcription Factor

HIF-1 (hypoxia inducible factor 1) performs a crucial role in mediating the response to hypoxia. However, other transcription factors are also capable of regulating hypoxia-induced target-gene transcription. In a previous report, we demonstrated that the transcription factor ATF-2 (activating transcription factor 2) regulates hypoxia-induced gene transcription, along with HIF-1α. In the present study, we show that the protein stability of ATF-2 is induced by hypoxia and the hypoxia-mimic CoCl2 (cobalt chloride), and that ATF-2 induction enhances HIF-1α protein stability via direct protein interaction. The knockdown of ATF-2 using small interfering RNA and translation-inhibition experiments demonstrated that ATF-2 plays a key role in the maintenance of the expression level and transcriptional activity of HIF-1α. Furthermore, we determined that ATF-2 interacts directly with HIF-1α both in vivo and in vitro and competes with the tumour suppressor protein p53 for HIF-1α binding. Collectively, these results show that protein stabilization of ATF-2 under hypoxic conditions is required for the induction of the protein stability and transactivation activity of HIF-1α for efficient hypoxia-associated gene expression.

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The Hypoxia-Inducible Factor 2α N-Terminal and C-Terminal Transactivation Domains Cooperate To Promote Renal Tumorigenesis In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.01514-06 ◽

2007 ◽

Vol 27 (6) ◽

pp. 2092-2102 ◽

Cited By ~ 122

Author(s):

Qin Yan ◽

Steven Bartz ◽

Mao Mao ◽

Lianjie Li ◽

William G. Kaelin

Keyword(s):

Transcriptional Activation ◽

Hypoxia Inducible Factor ◽

Gene Activation ◽

Tumor Formation ◽

Transactivation Domain ◽

Renal Carcinogenesis ◽

Von Hippel Lindau ◽

Transactivation Domains ◽

Relative Contribution

ABSTRACT Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor, consisting of an alpha subunit and a beta subunit, that controls cellular responses to hypoxia. HIFα contains two transcriptional activation domains called the N-terminal transactivation domain (NTAD) and the C-terminal transactivation domain (CTAD). HIFα is destabilized by prolyl hydroxylation catalyzed by EglN family members. In addition, CTAD function is inhibited by asparagine hydroxylation catalyzed by FIH1. Both hydroxylation reactions are linked to oxygen availability. The von Hippel-Lindau tumor suppressor protein (pVHL) is frequently mutated in kidney cancer and is part of the ubiquitin ligase complex that targets prolyl hydroxylated HIFα for destruction. Recent studies suggest that HIF2α plays an especially important role in promoting tumor formation by pVHL-defective renal carcinoma cells among the three HIFα paralogs. Here we dissected the relative contribution of the two HIF2α transactivation domains to hypoxic gene activation and renal carcinogenesis and investigated the regulation of the HIF2α CTAD by FIH1. We found that the HIF2α NTAD is capable of activating both artificial and naturally occurring HIF-responsive promoters in the absence of the CTAD. Moreover, we found that the HIF2α CTAD, in contrast to the HIF1α CTAD, is relatively resistant to the inhibitory effects of FIH1 under normoxic conditions and that, perhaps as a result, both the NTAD and CTAD cooperate to promote renal carcinogenesis in vivo.

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Hypoxia Rapidly Induces the Expression of Cardiomyogenic Factors in Human Adipose-Derived Adherent Stromal Cells

Journal of Clinical Medicine ◽

10.3390/jcm8081231 ◽

2019 ◽

Vol 8 (8) ◽

pp. 1231

Author(s):

Choi ◽

Moon ◽

Jung ◽

Lim ◽

Lee ◽

...

Keyword(s):

Stem Cells ◽

Stromal Cells ◽

Hypoxia Inducible Factor ◽

In Vivo Studies ◽

Hypoxic Stress ◽

Differentiation Markers ◽

Hypoxia Inducible Factor 1 ◽

Hypoxic Conditions ◽

Fibroblast Growth Factor 19

Background: The efficacy of interstitial vascular fraction (SVF) transplantation in the treatment of heart disease has been proven in a variety of in vivo studies. In a previous study, we found that bone marrow-derived mesenchymal stem cells (BM-MSCs) altered their expression of several cardiomyogenic factors under hypoxic conditions. Methods: We hypothesized that hypoxia may also induce obtained adipose-derived adherent stromal cells (ADASs) from SVFs and adipose-derived stem cells (ASCs) to differentiate into cardiomyocytes and/or cells with comparable phenotypes. We examined the differentiation markers of cell lineages in ADASs and ASCs according to time by hypoxic stress and found that only ADASs expressed cardiomyogenic markers within 24 hours under hypoxic conditions in association with the expression of hypoxia-inducible factor 1-α (HIF-1α). Results: Differentially secreted proteins in a conditioned medium (CM) from ASCs and ADASs under normoxic or hypoxic conditions were detected using an antibody assay and may be associated with a dramatic increase in the expression of cardiomyogenic markers in only ADASs. Furthermore, the cardiomyogenic factors were expressed more rapidly in ADASs than in ASCs under hypoxic conditions in association with the expression of HIF-1α, and angiogenin, fibroblast growth factor-19 (FGF-19) and/or macrophage inhibitory factor (MIF) are related. Conclusions: These results provide new insights into the applicability of ADASs preconditioned by hypoxic stress in cardiac diseases.

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Cellular and molecular mechanisms of hypoxia-inducible factor driven vascular remodeling

Thrombosis and Haemostasis ◽

10.1160/th06-12-0744 ◽

2007 ◽

Vol 97 (05) ◽

pp. 774-787 ◽

Cited By ~ 52

Author(s):

Norbert Weissmann ◽

Friedrich Grimminger ◽

Werner Seeger ◽

Frank Rose ◽

Jörg Hänze

Keyword(s):

Vascular Remodeling ◽

Molecular Mechanisms ◽

Target Genes ◽

Genetic Manipulation ◽

Hypoxia Inducible Factor ◽

Ischemic Disease ◽

Hypoxic Conditions ◽

Concomitant Reduction ◽

Adaptive Processes

SummaryHypoxia-inducible factor (HIF) is an oxygen-dependent transcription factor that activates a diverse set of target genes, the products of which are involved in adaptive processes to hypoxia. Employing genetic manipulation of HIF expression, in-vivo and cellular studies have focused on HIF as a crucial factor affecting hypoxia-induced vascular remodeling.Vascular remodeling comprises processes which establish and improve blood vessel supply such as vasculogenesis, angiogenesis and arteriogenesis. These processes are observed during ontogenesis, tumor progression, ischemic disease or physical training. Furthermore, under hypoxic conditions, a pulmonary-specific type of vascular remodeling called pulmonary arterial remodeling occurs that is characterized by thickening of the vessel wall with a concomitant reduction in the vessel lumen area, thereby limiting blood flow.This response results in pulmonary hypertension with right ventricular hypertrophy, a lethal disease. In this review, we summarize and discuss mechanisms by which HIF interferes with the different vascular remodeling processes.

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A Novel Role for Mesenchymal Stem/Progenitor Cells As Hypoxia Sensors During Initiation of Neo-Vasculogenesis in Vivo

Blood ◽

10.1182/blood.v120.21.613.613 ◽

2012 ◽

Vol 120 (21) ◽

pp. 613-613

Author(s):

Nicole A Hofmann ◽

Anna Ortner ◽

Rodrigo Jacamo ◽

Andreas Reinisch ◽

Katharina Schallmoser ◽

...

Keyword(s):

Progenitor Cells ◽

Ambient Air ◽

Culture Conditions ◽

Cell Phenotype ◽

Specific Chemical ◽

Vessel Formation ◽

Medical Needs ◽

Hypoxic Conditions ◽

Vascular Regeneration

Abstract Abstract 613 Background: Hypoxia is a major stimulus of neo-vasculogenesis. Under hypoxic conditions endothelial colony-forming progenitor cells (ECFCs) arrange tubular structures, which can connect to the pre-existing vasculature forming functional perfused vessels. The current view is that mesenchymal stem and progenitor cells (MSPCs) or their pericyte progeny are recruited subsequently to stabilize vessels. So far, clinical applications of endothelial progenitors to restore tissue oxygenation after ischemia, cardiovascular disease or stroke largely failed to meet medical needs. Based on previous work demonstrating patent vessel formation after MSPC/ECFC co-transplantation in vivo (Blood 2009), we hypothesized that MSPCs have a decisive role in the vasculogenic response to hypoxia. Here we show for the first time that ECFCs in hypoxic conditions in vivo strictly require the presence of functional MSPCs not only to stabilize but primarily to initiate neo-vasculogenesis by a hypoxia-inducible transcription factor (HIF)-dependent mechanism. Methods: Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. Progenitor cell phenotype, long-term proliferation, HIF stabilization, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O2), venous oxygen (5% O2) and standard ambient air culture conditions (20% O2). ECFC and MSPC crosstalk in vivo was studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelet lysate gel). Cell type-specific chemical and genetic inhibition of HIF (YC-1, shRNA) was used to delineate the role of hypoxia sensing in MSPCs and ECFCs, respectively, during vasculogenesis in vivo. To determine if downstream target proteins of HIF-1α could substitute for MSPC presence during vasculogenesis, selected growth factors and cytokines were tested. Results: Progenitor proliferation and function in vitro were reduced with declining oxygen levels. ECFCs stabilized hypoxia-inducible factor-1α (HIF-1α) only at 1% O2, while MSPCs stabilized HIF-1α already at 5% O2. In an NSG mouse model, ECFCs transplanted into a hypoxic environment did not stabilize HIF-1α, while transplanted sole MSPCs or MSPCs in co-transplants showed strong nuclear HIF-1α stabilization 1 day after transplantation preceding any vessel formation or perfusion. In the absence of MSPCs, the majority of ECFCs underwent apoptosis within 24h in vivo. Inhibition of HIF-1α stabilization in MSPCs but not in ECFCs significantly abrogated vessel formation in vivo. Blocking the prominent HIF-1α down-stream target vascular endothelial growth factor (VEGF) resulted in the expected inhibition of neo-vasculogenesis. Interestingly, substitution of VEGF alone could not restore vessel formation, neither when injected together with sole ECFCs nor in a model where ECFCs were co-transplanted with HIF-depleted MSPCs. Substitution of a complex mixture of platelet-derived factors in vivo partly restored the vasculogenic function of HIF-depleted MSPCs. Conclusions: MSPCs react to a low oxygen environment by stabilizing HIF-1α earlier and more sensitively than ECFCs. MSPCs promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis in the initial phase of vasculogenesis by a HIF-dependent trophic mechanism. Surprisingly, therapeutic vasculogenesis can occur independently of endothelial HIF stabilization. These results argue in favor of MSPC/ECFC co-transplantation as a promising strategy for vascular regenerative therapy. The observation that VEGF alone could not compensate for the vasculogenic competence of pericyte precursors in vivo underlines the complexity of the hypoxia-induced cytokine network. The fact that hypoxia sensing in MSPCs but not in ECFCs is crucial to initiate vascular regeneration supports a shift of focus from endothelial cells to perivascular mesenchymal cells as a therapeutic target in anti-angiogenic therapy. Disclosures: No relevant conflicts of interest to declare.

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EBV-Encoded Mirnas Facilitate Inflammation and Tumor Formation Through Both Intra- and Inter-Cellular Regulation In Vivo Mouse Model

Blood ◽

10.1182/blood.v122.21.3735.3735 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3735-3735

Author(s):

Natsuko Yamakawa ◽

Jun Ogata ◽

Takashi Yahata ◽

Jun Lu ◽

Kazuaki Yokoyama ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Cells ◽

Small Rnas ◽

Conflicts Of Interest ◽

Functional Characterization ◽

Tumor Formation ◽

Sequencing Analysis ◽

Immunodeficient Mice

Abstract Introduction EB virus (EBV) is associated with heterogeneous lymphomas. Hodgkin's lymphoma (HL) cells are embedded in non-neoplastic bystanders: B, T cells, and macrophages. Without these bystander cells, the lymphoma cells are incapable of being engrafted in immunodeficient mice. In this context, the bystanders are tumor-supportive “inflammatory niche”. Recently, EBV-infected cells produce exosomes that contain EBV specifically encoded miRNAs (EBV-miRNAs). The miRNAs are transferred to cells, and involved in tumor metastasis. However, the detailed mechanism is unknown. Accordingly, we hypothesized that exosomal EBV-miRNAs might redirect tumor surrounding immune cells from tumor reactive into tumor-supportive “inflammatory niche”. Methods We evaluated the expression of EBV-miRNAs in EBV+HL clinical specimens by in situ hybridization, their functional characterization in vitro, and their effects on persistent infection and tumor development in vivo humanized NOG mice model. Moreover, in order to clarify its sorting mechanism, trans factor and cis factor which determined secreted and non-secreted miRNAs was analyzed by use of mass-spectrograhy and next-generation sequencing. Results and Discussion The EBV-miRNAs effects were potent on monocyte/macrophage Mo/Mf in inducing CD69, IL-10, and TNF, suggesting that EBV-miRNAs might polarize Mo/Mf into tumor associated Mf (TAM). EBV-miRNAs suppress tumor cell proliferation in vitro, implying that it works as tumor-suppressor in the tumor cells, while they are required to develop LPD in vivo, which seems contradict to the result in vitro. These results suggest that EBV-miRNAs intra-cellularly regulate the tumor cells to adjust to the surrounding circumstances, for example, to escape from immune surveillance, and inter-cellularly regulate Mo/Mf to support the tumor survival or development. Most importantly, exosomal EBV-miRNAs derived from the tumor cells were transferred to Mf in human EBV+ HL samples. Interestingly, one EBV coded miRNA was not secreted at all, though it abundantly expresses in the cells. The miRNA has been reported to strongly promote cell proliferation in EBV infected tumor cells. It made us hypothesized that the sorting system of secretary and non-secretary miRNAs is critical in the formation of “inflammatory niche”. In order to clarify the mechanism of the sorting, the chimeric miRNA was constructed then, we determined the sequence, which regulates secretion and non-secretion, and purified the protein complex, which specifically bound to the sequence. Mass spectrography and successive knockdown assay, the trans factor which inhibits secretion was identified. Moreover, the next sequencing analysis for the small RNAs revealed that abundant EBV-coded small RNAs occupied RNA-induced silencing complex (RISC), and that non-secreted EBV-miRNA was specifically modified. It is now under investigation whether the modification is involved in the sort mechanism between secretary and non-secretary miRNAs. Taken together, EBV-miRNAs have critical roles in intra- and inter-cellular manner. Especially, the functions as an inter-cellular communicator might be important in the tumor formation and the mechanism needs further investigation. Disclosures: No relevant conflicts of interest to declare.

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