Signaling via the B cell receptor (BCR) is essential for B cell survival and development, and antibody production in both physiological and pathological conditions. The nature of BCR signaling is varied in different subpopulations and developmental stages of B cells and can be classified into tonic, chronic active, and priming signaling , 2. WherMeyer et al. reviews Siglecs in B cells, highlighting the interplays between CD45 and CD22 (Siglec-2), CD22 and Galectin-9, and their influence on BCR signaling. Both CD22 and Siglec-G contain immune receptor tyrosine-based inhibitory motifs (ITIMs) within their cytoplasmic tails and recruit the tyrosine phosphatase SHP1, which inhibits B cell signaling. Whereas, CD22 mainly functions in conventional B cells, Siglec-G inhibit the constitutive BCR signaling in autoreactive B-1a cells, probably by binding in cis to the Cμ1 domain of BCR-IgM in the steady-state through α2,3- or α2,6 linked sialic acids  family play important roles in regulating BCR signaling. Rip et al. show that the high level of BTK expression in B cells enhances their sensitivity to TLR stimulation, suggesting that BTK promotes the synergistic activation of BCR and TLR engagements. In another direction, Maity et al. discusses how the cross-talks between chemokine receptor CXCR4 and the BCR of different immunoglobulin isotypes control the development and survival of leukemic B cells.B cell activation and differentiation is also critically dependent on signaling through pattern recognition receptors and chemokine receptors. Although BCR-mediated signaling pathways are largely distinct from Toll-like receptor (TLR) signaling pathways, these two pathways interact through shared signaling molecules, such as STAT3 activation by DOCK8-MyD88-Pyk2 complex . TLR-medLi et al. review the coordination between the actin cytoskeleton and BCR signaling, highlighting the potential role of actin in the initiation of BCR triggering. Actin remodeling is also important for the antigen extraction at the immunological synapse (IS) of B cells. Ibañez-Vega et al. show that proteasome activity is required for the dispersion of actin at the centrosome, which is important for lysosome recruitment to the IS, antigen extraction, and antigen presentation. Germinal center (GC) B cells extract antigen using multiple small peripheral BCR clusters instead of a single large IS seen in naïve or memory B cells. Actin remodeling is very critical for antigen extraction by GC B cells, relating actin-generated extraction forces to selection of high affinity GC B cells. The actin regulators during the GC response are reviewed by He and Westerberg.The BCR has also a close relationship with the actin cytoskeleton. BCR activation leads to reorganization of the cortical actin and conversely, changes in the actin cytoskeleton influence BCR signaling. Alsufyani et al. show the role of Mst1 and Mst2, mammalian orthologs of Hippo proteins, in B cell migration and homing. Follicular B cells lacking both Mst1 and Mst2 were shown to be unable to recirculate beyond spleen. It is also likely that Mst1 is required for migration of precursors of B-1a cells as B-1a cell development was defective in Mst1−/− mice. Focusing on IgM deficiency in patients with novel mutations in BTK and BLNK, Geier et al. show that even hypomorphic mutations in these genes can lead to impaired B cell homeostasis.Regulation of migration critically determines B cell development and functions. In this topic, Ma et al., accumulate with aging and show altered BCR repertoire. ABCs may play a significant role in autoimmune diseases by secreting autoreactive Abs and inducing Th17 cell differentiation. Ambegaonkar et al. shows that T-bet+ ABCs are induced by prolonged antigen stimulation in the presence of CpG and IFNγ.With aging, bone marrow B cell production is inefficient and altered B cell homeostasis leads to expansion of activated B cells with atypical characteristics such as the expression of CD11b, CD11c, and T-bet. These B cells, termed as age-associated B cells (ABCs) and reviewed by We thank all the authors and reviewers for their invaluable work, which made the publication of this Research Topic possible. We hope that this collection of articles will serve as a useful source of knowledge to those interested in B cell activation and pathology.TK made a substantial contribution when drafting this editorial essay. All authors listed have made direct intellectual contributions to the work, and approved it for its publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The protein tyrosine phosphatase interacting protein 51 (PTPIP51) regulates and interconnects signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway and an abundance of different others, e.g., Akt signaling, NF-κB signaling, and the communication between different cell organelles. PTPIP51 acts as a scaffold protein for signaling proteins, e.g., Raf-1, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), as well as for other scaffold proteins, e.g., 14-3-3 proteins. These interactions are governed by the phosphorylation of serine and tyrosine residues of PTPIP51. The phosphorylation status is finely tuned by receptor tyrosine kinases , non-receptor tyrosine kinases (c-Src) and the phosphatase protein tyrosine phosphatase 1B (PTP1B). This review addresses various diseases which display at least one alteration in these enzymes regulating PTPIP51-interactions. The objective of this review is to summarize the knowledge of the MAPK-related interactome of PTPIP51 for several tumor entities and metabolic disorders. RSK, mitogen-activated protein kinase interacting protein kinases 1 and 2 (MNK1/2), Ets, Ets domain-containing protein (Elk1), Myc, signal transducer and activator of transcription 1/3 (STAT1/3) and estrogen receptor (ER), to name some of them [The mitogen-activated protein kinase (MAPK) pathway is one of the best described signaling system in cancer. Almost one third of all human cancers have reported alterations in MAPK signaling, indicating the high relevance of the precise understanding of this pathway . The basSince, the MAPK pathway controls these essential functions a precise regulation and titration of the signaling activity is needed. A perfect example for such a strict regulation is the upstream positioned Raf kinases. The Raf kinase family consists of three different Raf proteins, Raf-1, B-Raf and A-Raf. Although their structures are almost similar, their activation modes are extremely different. After recruitment of Raf kinases to GTP-bound Ras, a complex series of phosphorylations is induced for activation. These phosphorylations are needed for the activation of the kinase domain and reduction of the autoinhibition. The activation of Raf-1 and A-Raf requires phosphorylation of the N-region, dephosphorylation of the S259 inhibitory site, and phosphorylation of the activation loop. B-Raf is already in a preactivated state and can be fully activated by Ras alone, whereas the activation of Raf-1 and A-Raf requires other factors [Another regulating mode of the Raf kinases is the binding to scaffolding proteins such as the 14-3-3 protein family . Such adOf note, the protein tyrosine phosphatase interacting protein 51 (PTPIP51) represents another scaffold protein, which regulates MAPK activation on Raf-1 level . PTPIP51Besides the direct regulation of the MAPK pathway, PTPIP51 is involved in a broad range of cellular functions and signaling systems. The panel of interaction partners ranges from NF-κB signaling proteins  over mitochondrial associated ER membrane-related proteins , autophagy-related signaling, and mitosis associated proteins  ,18,19,20Obesity and insulin resistance and their secondary diseases have reached epidemic proportions. More than one third of the US population has a diagnosed obesity and 10% of the adults are affected of diabetes . The annThe most important signaling pathways for the regulation of lipid storages in adipocytes are the insulin receptor and the PKA signaling, which act as antagonists . WhereasThe activity level of the insulin receptor is not only determined by the presence of insulin, but also by the phosphorylation status of the receptor. The phosphatase PTP1B represents a crucial downregulator of insulin receptor activity ,27. The The expression of PTPIP51 in adipocytes is tightly regulated by the diet and the submission to training in mice . Bobrichhttp://gps.biocuckoo.org/), PKA was identified to phosphorylate the serine residue 149 of PTPIP51 [PTPIP51 also interacts with the insulin receptor signaling antagonist, the PKA ,9. PKA i PTPIP51 ,9,11. HeThe central position in metabolic signaling mirrors the important role of PTPIP51 in the genesis of insulin resistance and obesity. Up to now, PTPIP51 seems to maintain a level of insulin sensitivity via stimulation of MAPK signaling. As mentioned above this stimulating effect is active if PTPIP51 is dephosphorylated at the Tyr176 residue. This conflicts with the observed outstanding findings of PTP1B knockout mice, since PTP1B is essential for the dephosphorylation of PTPIP51 ,10,11,12Breast cancer is the most common neoplasm in women, accounting for about 25% of all diagnosed tumors. Although early diagnosis and enhanced therapies of breast carcinomas greatly improved the overall survival time, breast cancer is still the third most cause of cancer deaths in the US ,32.About one third of the breast tumors exhibit an overexpression of the HER2/ErbB2 receptor, leading to a more aggressive and invasive growth of the cancer cells and thus an impaired overall survival time . The ampThe knowledge about Her2 receptor amplification and its functional consequences in breast tumors led to the development of several Her2 targeted therapies encompassing small molecule tyrosine kinase inhibitors and monoclonal antibodies ,36. IntrDespite the good clinical results, the management of anti Her2 therapy resistances are still challenging. Several signaling pathways have been identified to mediate these resistances, e.g., the PI3K-Akt-mTOR signaling, c-MET signaling pathway or low immune response ,39,40.The regulation of the non-receptor tyrosine kinase c-Src depicts another crucial resistance mechanism ,42. c-SrFor the precise titration of phosphorylation levels of receptor tyrosine kinases (RTKs), signaling kinases and scaffolding proteins not only kinases, such as c-Src, are needed but also phosphatases. One of the best described phosphatases is the protein tyrosine phosphatase 1B (PTP1B) . PTP1B iThe scaffolding protein PTPIP51 represents a crucial crossing point of all the aforementioned tumor promoting and resistance inducing mechanisms. PTPIP51 is expressed in normal breast glands as well as in breast cancer cells, whereby the expression of PTPIP51 protein is diminished in the cancer cells (ongoing studies of our group). Further analysis of the crucial Tyr 176 phosphorylation site of PTPIP51 showed a strong upregulation of the phosphorylation in breast cancer cells. As shown by Brobeil et al. the Tyr 176 residue phosphorylation regulates the binding of PTPIP51 to Raf-1 via 14-3-3β and thereby exerts its MAPK stimulating effect . The dowIn Her2 amplified breast cancer cells, the phosphorylation of PTPIP51 at Tyr176 is to a great extend performed by the EGFR. Inhibition of the EGFR in Her2 amplified breast cancer cells induces a reduction of PTPIP51 phosphorylation at the Tyr176 residue accompanied by a formation of the Raf-1/14-3-3β/PTPIP51 interactome, thus proofing a normal regulation of MAPK-related interactions of PTPIP51 .Interestingly, PTPIP51 also interacts with the Her2 receptor, but it is not clear if the Her2 receptor phosphorylates PTPIP51 or if PTPIP51 forms a scaffold for the interaction of the Her2 receptor with other signaling molecules . Of noteIn addition, own studies showed the relevance of the PTPIP51/c-Src interaction in correlation with the sensitivity of Her2 positive breast cancer cells to EGFR/Her2 targeted TKIs. Application of several EGFR/Her2 targeted TKIs to the Her2 amplified breast cancer cell line SK-BR3 led to a highly significant augmentation of PTPIP51/c-Src interaction, whereas the same treatment of BT474 cells, also a Her2 amplified breast cancer cell line, did not alter or even reduce the PTPIP51/c-Src interaction. Interestingly, the enhanced interaction of PTPIP51 and c-Src was accompanied by a less sensitivity to TKI treatment, representing another potential resistance mechanism .Of note, as aforementioned the interaction of PTPIP51 and c-Src is severely altered in Her2 amplified breast cancer cells when treated with EGFR/Her2 targeted TKIs, but the interaction of PTPIP51 and its crucial phosphatase PTP1B remains nearly unaffected . Solely Additionally, the importance of the PTPIP51/PTP1B interaction is underlined by the fact, that this interaction is directly correlated with the grading of breast carcinomas. Ongoing studies of our group examined the interaction of PTPIP51 and PTP1B in breast carcinomas of no special type and showed a significant enhancement of the interaction in grade 3 carcinomas compared to grade 1 and 2 carcinomas. Moreover, Her2 amplified carcinomas also displayed a significantly upregulated PTPIP51/PTP1B interaction compared to Her2 negative breast cancer samples. This may be supported by the upregulation of PTP1B in Her2 amplified breast cancer.To sum up, PTPIP51 is needed for the normal function of healthy mammary glands and its expression is altered in the development of breast tumors. Furthermore, the interaction of PTPIP51 and PTP1B correlates with the grading and the Her2 amplification, indicating an alteration of PTPIP51 phosphorylation during the progression of breast carcinoma. We were able to unveil a potential role of PTPIP51 in tumor promoting signaling and therapy resistance against EGFR/Her2 targeted TKIs mediated through the non-receptor kinase c-Src and the phosphatase PTP1B . In consOne of the most malignant tumors with an overall survival time of about one year, despite significant advances in the therapeutic options, is the glioblastoma multiforme (GBM) ,52,53. TConsequently, many GBMs must have an intrinsic or a rapidly acquired resistance against EGFR inhibitors or the signaling is bypassed via alternative growth and survival promoting pathways . TherefoRSK, MNK1/2, Ets, Elk1, Myc, STAT1/3, which mediate the proliferation and growth inducing effects of the EGFRvIII activation [The most commonly activated signaling pathway downstream of the EGFRvIII is the ERK signaling . The acttivation ,7. 14-3-tivation . Therebytivation . Furthertivation . Thorsontivation .In the setting of GBM 14-3-3 proteins also seem to play a crucial role. Yang and coworkers showed a direct correlation between the amount of expressed 14-3-3β and 14-3-3η and the grade of malignancy of glioma . The impThe examination of the aforementioned cellular signaling and many more, e.g., platelet-derived growth factor receptor (PDGFR) signaling, neurofibromatosis type 1 (NF1) and the tumor microenvironment, lead to crucial insights in the aberrant tumor signaling, but up to now a linkage between these structures is still missing.PTPIP51 depicts a possible functional linkage between the constitutively activated EGFRvIII and the scaffolding protein 14-3-3β. PTPIP51 is expressed in glioma of low malignancy as well as in GBM . The expInterestingly, the PTPIP51 protein structure includes two conserved regions, which facilitate the binding to 14-3-3 proteins ,9. MoreoAs aforementioned, the inhibition of the constitutively activated EGFRvIII seemed a promising therapeutic strategy, but up to now the clinical results lack a sufficient improvement of the patients’ outcome . Intereshttps://seer.cancer.gov/statfacts/html/melan.html). From all cancers melanomas have the highest probability to metastasize to the brain [Melanomas belong to a cancer entity arising from melanocytes originally derived from neural crest cells found in skin and uvea. Among the three main types of skin cancer, beside melanoma there are basal cell carcinoma and squamous cell carcinoma. The melanomas account for about 2% of skin cancers, but for 75% to 90% of deaths . Melanomhe brain . In 50% he brain .Neurofibromin 1 (NF1) mutated (14%) and in “triple wild type” [For the pathogenesis of melanomas, the activation of the MAPK pathway is essential . This isld type” . The knoCRAF/Raf-1 is expressed in all cells, but BRAF is associated with cells of neuronal origin such as melanocytes. CRAF mutations are rare in contrast to BRAF mutations . BRAF muFor example, vemurafinib treated patients showed an overall response rate of 52.2% and the median progression-free survival was 8.3 months; the median overall survival was 13.5 months according to a study of Si and coworkers . This foMechanisms promoting resistances can be manifold: a reactivation of MAPK signaling (amplification or activation of target kinases); bypass via different signaling pathways, e.g., PI3K-Akt-mTOR signaling; or by the surrounding stromal cells, which secrete HGF in BRAFi-resistant melanomas, activating MAPK and PI3K-Akt-mTOR.Next to the more common BRAF oncogene, mutations in the NRAS gene result in more aggressive melanomas . BRAF muNRAS mutations lead to a substitution of the amino acids at positions G12 or G13 making NRAS insensitive to the inactivation by the Ras GTPase-activating proteins, or more common at position Q61, where Q61R is predominant in melanoma cells . Ras mutAs there exist no specific NRAS/Ras inhibitors, different strategies for treatment of NRAS-mutated melanomas were administered for therapy. The use of farnesyl transferase inhibitors to prevent posttranslational modification of Ras and its insertion into the plasma membrane failed in clinical studies . Third gThe first immunotherapies successfully approved interleukin 2 and anti-CTLA4 antibody both stimulating the immune response as treatment options . As seenPTPIP51 protein and its interactome in melanoma cells in relation to normal melanocytes derived from common nevi, as well as to melanocytes from dysplastic nevi revealed a characteristic profile within the three different entities. Dysplastic nevi represent an intermediate state between nevi and melanomas as they are morphologically and biologically intermediate between these two entities .Despite massive changes in the molecular characteristics of melanocytes from normal nevi to dysplastic nevi and further on to malignant melanocytes, there is no significant change in the amount of PTPIP51 protein in these different stages. Yet, if the grade of Tyrosine 176 phosphorylation is analyzed, melanocytes from dysplastic nevi displayed the highest level in Tyr 176 phosphorylated PTPIP51 compared to the Tyr176 phosphorylation status of melanocytes derived from normal nevi, exhibiting the lowest phosphorylation level and when compared to the phosphorylation state in melanocytes from malignant melanomas displaying a tyrosine phosphorylation level laying in between. These findings are corroborated by the interaction profile of PTPIP51 and PTP1B, the phosphatase which is responsible for dephosphorylation of tyrosine 176, with the highest number of interactions in normal melanocytes and lowest number of interactions in melanocytes from dysplastic nevi and an interaction profile laying in between the aforementioned for malignant melanocytes. As described above, Tyr176 phosphorylation regulates the interaction of PTPIP51 with Raf. Solely when dephosphorylated at Tyr176 PTPIP51 can bind to Raf or 14-3-3β protein promoting MAPK signaling.PTPIP51 affects the MAPK pathway by interacting with Raf-1 to stimulate downstream signaling. In melanocytes the interaction with the Raf-1/CRAF protein is relatively low. Nevertheless, there are quantitative differences for the three entities with lowest interaction numbers in malignant melanocytes and highest numbers in melanocytes from dysplastic nevi. Noteworthy, the interaction with BRAF is up to 100 times more numerous in the three different entities. PTPIP51/BRAF interaction is lowest in dysplastic nevi melanocytes, and somewhat higher in melanoma cells and highest in healthy melanocytes, where the number of interactions was five times higher compared to that of dysplastic nevi cells. This pattern corresponds well to that seen for PTPIP51 and PTP1B interaction and the measured phosphorylation status of PTPIP51. In addition, as indicated by the interaction profile of PTPIP51 and PKA, serine 212 phosphorylation which enhances the interaction of PTPIP51 and Raf, is much more reduced in dysplastic nevi and to a lesser degree in malignant melanoma cells as indicated by their lower interaction levels of PTPIP51/PKA in comparison to an almost doubled interaction seen in normal melanocytes. Noteworthy, serine212 phosphorylation promotes the activation of MAPK signaling by augmenting the interactome of PTPIP51, 14-3-3β and Raf-1.These data argue for a counter-regulatory function of PTPIP51 in dedifferentiating melanocytes trying to reduce the stimulation by PTPIP51 interaction on BRAF level displaying lowest levels in dysplastic nevi and somewhat higher levels in malignant melanomas. The relative rise in the number of interactions in malignant melanocytes probably reflects the ongoing further dedifferentiation of the cells, thus leading to an increased MAPK signaling compared to dysplastic nevi and the highest stimulation to proliferate.BRAF inhibitor resistance of melanoma can be a consequence of bypassing MAPK signaling via the PI3K-Akt pathway. Noteworthy, PTPIP51 is also involved in Akt signaling via a direct interaction with Akt protein. In untreated samples either from healthy control nevi or dysplastic nevi or from melanoma, melanocytes of the control display a high PTPIP51/Akt interaction, which is strongly reduced in samples from dysplastic nevi or melanoma. Akt stabilizes the communication site between mitochondria and endoplasmic reticulum. PTPIP51 likewise interferes in this mitochondria-ER relation via its VAPB interaction. The communication between both organelles is among other factors necessary for apoptosis . PresumaTo overcome therapy resistance in melanoma MEK inhibitors are tested in combination with a variety of drugs that use different approaches: inhibition of upstream Ras effectors, inhibition of PI3K-Akt-mTOR, inhibition of cell cycle regulators and activation of anti-tumor immunity but all seem to fail according to the existing cross-resistances .This strongly emphasizes the need for a better understanding of the MAPK/PTPIP51 interactome in melanoma A.Leukemias are a group of heterogenous diseases with highly different malignant potential. In this context, two entities must be discriminated: acute leukemias and chronic leukemias. The later encompasses the chronic lymphocytic leukemia, which is classified as a non-Hodgkin lymphoma, and the chronic myelogenous leukemia. Both diseases are not curable but exhibit a prolonged course, e.g., with an eight years survival time of almost 87% for the chronic myeloid leukemia .Acute leukemias are also subdivided into the two cell lineages, lymphocytic and myelogenous. Here, the group of acute myeloid leukemia (AML) is a highly malignant neoplasm exhibiting a near stable incidence over the last years with 3.7 affections per 100,000 persons and an age-dependent mortality of 2.7 to nearly 18 per 100,000 persons. The disease continuously shows two peaks in early childhood and later adulthood . MoreoveInterestingly, the prognosis as well as therapeutic decisions are tightly linked to specific cytogenetic and molecular alterations within the malignant transformed cells , encompaThe FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase which is expressed on the surface of CD34+ hematopoietic stem cells and other immature hematopoietic progenitors.The receptor is classified as a type-1 transmembrane receptor tyrosine kinase encompassing several functional domains, e.g., an extracellular domain with Ig domains, TMD, and an intracellular tyrosine kinase domain with two kinase domains . FLT3 isApproximately 30% of AML patients harbor some form of FLT3 mutation, which can be divided itself into two mutational classes: internal tandem duplications (FLT3/ITD mutations) in or near the juxtamembrane domain of the receptor and point mutations resulting in single amino acid substitutions involving the activation loop of the tyrosine kinase domain (FLT3/TKD mutations) . Yet, thBesides the activation of whole signaling cascades, FLT3 is also able to phosphorylate specific signaling molecules, e.g., the Src family kinase Lyn. Compared to the wild type FLT3 receptor, FLT3/ITD displayed a higher affinity to bind to Lyn and the affinity was relative to the intensity of tyrosin phosphorylation of the receptor . Lyn is The other class III receptor tyrosine kinase, c-KIT, which also plays a pivotal role in AML, is expressed by myeloblasts in about 60% to 80% of patients: Therefore, next to the described FLT3 aberrations c-KIT harbors the most frequently observed activating RTK mutations in AML with an overall incidence of 17%. The mutations also encompass, for example, internal tandem duplications in c-Kit . The couPTPIP51 is expressed in malignant transformed blasts of AML in an isotype specific manner. PTPIP51 protein isoforms could be traced with molecular weights of 13 kDa, 25 kDa, 38 and 52 kDa, respectively . Using pAs Dasatinib blocks Lyn activity with consecutive apoptosis in imatinib-resistant CML cells , the intIn summary, PTPIP51 displays a disease-related isoform expression in AML with loss of functional domains. Yet, the physiological regulation of the MAPK binding capacity of PTPIP51 seems to be intact. Thus, the involved signaling molecules of PTPIP51 can be directly targeted by small molecules to induce apoptosis in AML blasts B.Protein-protein interactions are the foundation of all signaling events in normal cells as well as in dysregulated tumor cells. The framework for the correct procedure of signaling, the interconnection of different pathways and the appropriate subcellular localization is provided by scaffold proteins .In this review, we highlight the central position of PTPIP51 within the dysregulated MAPK pathway signaling of several disparate diseases. The interactions and thus the functions of PTPIP51 are regulated by the serine and tyrosine phosphorylation status of PTPIP51 ,10,11,12During the progression of tumorous diseases, the PTPIP51 regulating kinases and phosphatases often succumb alterations, in the form of activating mutations, overexpression or over-activation. C-Src represents a perfect example for tumor promoting and therapy resistance inducing kinases, which also regulates the phosphorylation of PTPIP51 ,41,46,67To sum up, PTPIP51 modulates the upmost position of the ERK pathway, the MAPKKK Raf-1. Furthermore, PTPIP51 crosslinks this signaling node with several tumor relevant RTKs, non-receptor tyrosine kinases and protein tyrosine phosphatases. PTPIP51 connects the MAPK pathway with several other signaling systems, e.g., the Akt and NF-κB signaling, which exceed the scope of this review. The precise understanding of the PTPIP51-related interactome in tumors is of the utmost interest and offers the possibility to understand dysregulated signaling systems and potential targetable signaling molecules. In particular, the interaction of PTPIP51 and c-Src seems to be of great relevance for therapy resistance mechanisms in several tumor entities and needs further investigation.


The interaction between the short 88Ser-Arg-Ser-Arg-Tyr92 sequence of the urokinase receptor (uPAR) and the formyl peptide receptor type 1 (FPR1) elicits cell migration. We generated the Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-NH2 (RI-3) peptide which inhibits the uPAR/FPR1 interaction, reducing migration of FPR1 expressing cells toward N-formyl-methionyl-leucyl-phenylalanine (fMLF) and Ser-Arg-Ser-Arg-Tyr (SRSRY) peptides. To understand the structural basis of the RI-3 inhibitory effects, the FPR1/fMLF, FPR1/SRSRY and FPR1/RI-3 complexes were modeled and analyzed, focusing on the binding pocket of FPR1 and the interaction between the amino acids that signal to the FPR1 C-terminal loop. We found that RI-3 shares the same binding site of fMLF and SRSRY on FPR1. However, while fMLF and SRSRY display the same agonist activation signature , translating binding into signaling, RI-3 does not interact with the activation region of FPR1 and hence does not activate signaling. Indeed, fluorescein-conjugated RI-3 prevents either fMLF and SRSRY uptake on FPR1 without triggering FPR1 internalization and cell motility in the absence of any stimulus. Collectively, our data show that RI-3 is a true FPR1 antagonist and suggest a pharmacophore model useful for development of compounds that selectively inhibit the uPAR-triggered, FPR1-mediated cell migration. When cell migration is deregulated, it contributes to a variety of pathologic conditions which include chronic inflammation, vascular diseases, and tumor metastasis3. For this reason, the control of cell motility may be useful for developing new therapeutic strategies aimed to control diseases driven by aberrant cell motility, such as the metastatic dissemination of cancer cells.Cell migration is a sequential and interrelated multistep process that regulates physiological processes such as embryonic development, tissue repair and immune-cell trafficking in both embryo and adult tissues4. Besides focusing proteolytic activity of uPA on the cell membrane5, uPAR, upon binding to uPA, initiates in a protease independent manner the intracellular signaling pathways that regulate physiologic processes such as wound repair and immune responses, as well as pathologic conditions such as inflammation and tumor metastases6.The Urokinase-type Plasminogen Activator (uPA) Receptor (uPAR) is a widely recognized master regulator of cell migration9. In the uPAR/uPA complexes, the domain boundary between DI and DII (uPAR84-95 sequence) is mostly projected on the external uPAR surface, comprises a protease-sensitive signaling region and exhibits a structural flexibility in both membrane-associated and soluble forms of uPAR11.uPAR is formed by three domains  linked by short sequence regions, anchored to the cell surface by a glycosyl-phosphatidylinositol (GPI) tail. The three domains pack together into a concave structure that binds uPAin vitro and in vivo14. We proven that both uPAR84-95 and SRSRY exert chemotactic activity by interacting with the formyl peptide receptor type 1 (FPR1) which, in turn, internalizes. After internalization, FPR1 triggers vitronectin receptor activation with an inside-out type of mechanism that involves PKC, AKT and MAPK14.We and others documented that the uPAR84-95 sequence as well as the synthetic shorter pentapeptide uPAR88-92 elicit chemotaxis and promote directional cell migration and angiogenesis 15, and identified Ser90 as a key residue within a critical “hinge”, where it affects the conformation of neighboring residues. Substitution of the Ser90 with a glutamic acid residue, in the membrane-associated uPAR, generated a dominant-negative protein that prevents agonist-triggered FPR1 internalization, migration and invasion of sarcoma cells16. Interestingly, a cyclized form of the SRSRY peptide elicits opposite effects on cell migration. The cyclic [SRSRY] peptide competes with both SRSRY and N-formylmethionyl-leucyl-phenylalanine (fMLF) peptides for binding to FPR1 and inhibits FPR1activation and internalization, causing a dramatic reduction of monocyte recruitment into inflamedtissues19.Using SRSRY peptide analogues, we found that the Arg89-Ser-Arg91 central core is indispensable for the SRSRY-dependent cell signaling21. Upon binding to formyl-peptide ligands, FPR1 elicits many responses including actin polarization, cell motility, production of reactive-oxygen species and release of cytokines by activating p38MAPK and PI3K/AKT signaling cascades23. Recently, FPR1 has been documented to be expressed also in non-myeloid cells, and many studies show that increased expression of FPR1 correlates with a poor prognosis in tumors of different origin29.Originally identified on the surface of myeloid cells as the high-affinity receptor for the fMLF peptide, FPR1 recognizes many and diverse ligands, ranging from formylated peptides derived from bacterial protein degradation and from mitochondrial proteins of eukaryotic cells, to non-formylated proteins/peptides and small compounds32. Although the proof-of-principle for this strategy was provided, none of these peptides revealed to be an ideal lead molecule since some of them are unstable to enzymatic digestion in human serum, which limits their half-life in vivo31, whereas the potent anti-angiogenic peptide Ac-L-Arg-Aib-L-Arg-D-Ca(Me)Phe-NH2 (UPARANT), which is stable in blood and reveals prolonged resistance to enzymatic proteolysis32, elicited some toxicity when systemically administered (unpublished).Some years ago, we generated libraries of uPAR-derived synthetic linear penta- and tetra-peptides, and peptidomimetic analogs carrying the Ser90 substituted with a glutamic acid residue or with the Cα-methyl-α-aminoisobutyric acid (Aib) and lead compounds were selected for their ability to inhibit uPAR/FPR1 interaction and reduce to basal levels cell migration and angiogenesis33. RI-3 is stable in human serum, adopts the turned structure typical of uPAR/FPR1 antagonists and competes with fMLF and SRSRY for binding to FPR1. RI-3 prevents fMLF-induced FPR1 internalization as well as p38 MAPK and PI3K/AKT signaling cascades, which are documented to mediate fMLF-triggered signal transduction pathways35. Furthermore, in the absence of any stimulus, RI-3 did not change the phosphorylation of p38 MAPK and PI3K/AKT protein kinases33.To overcome these liabilities, we applied the Retro-Inverso (RI) approach to these uPAR/FPR1 inhibitors. The retro-inverso Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-NH2 (RI-3) peptide was selected as the best inhibitor of the uPAR-mediated FPR1-dependent cell migrationThe pattern of activities of RI-3 prompted us to investigate the structure-function relationship between FPR1 and the fMLF, SRSRY and RI-3 peptides. This paper was organized according to the following objectives. The first one implied the molecular modeling of FPR1. The second objective was to study the structure-flexibility-function relationships of FPR1/fMLF and FPR1/SRSRY complexes by Molecular Dynamics (MD) and molecular docking simulations. Finally, we attempted to model and analyze FPR1/RI-3 complex in order to correlate RI-3 biological activity with the network of interaction between amino acids that signal to FPR1 C-terminal loop upon RI-3 engagement.36. In the Supplementary Fig. The 3D structure of the N-terminal and trans-membrane regions of FPR1 was modeled using comparative modeling and, as template, the structure of type-1 angiotensin II receptor  because it showed the highest sequence identity (32%) with the FPR1 sequence by Blast searchab initio approach of Quark server37, and selected the best model on the basis of TM-score, and energetic and stereo-chemical quality as described in our recent paper38. The best elected model had a TM-score equal to 0.37, an energetic Z-score of −4.28, and 94.3% of the residues in the allowed regions in the Ramachandran Plot. Finally, the complete structural model of FPR1 was obtained by comparative modeling, using as templates the obtained models for N-terminal and trans-membrane regions and for C-terminal domain. The final structure had 96.8% of the residues in allowed regions in the Ramachandran Plot, and an energetic score of −3.28. As shown in Fig. 39. FPR1 presents a well-defined binding pocket characterized by positively charged residues like Arg84 and Lys85 located in TM2 and Arg201 and Arg205 on TM5, and by the negatively charged residues Asp284 on TM7and Asp106 on TM3. However, between these two zones there are also hydrophobic residues that separate these charged areas and interact with the ligands. In detail, from one side there are Phe81 on TM2 and Phe102 on TM3 that form the anchor region with Arg84 and Lys85 located always on TM2 whereas from the other side there are Tyr257 on TM6 and Phe291 on TM7 that form the activation region with Arg201 and Arg205 located on TM5  the binding site, that is “cone-shaped with the tip towards the trans-membrane region”, is small and can accommodate only one peptide at a time considering that its radius (of about 8 Å) and its height (of about 13 Å) are lower than the length of the peptides (of about 14 Å); ii) the charged and aromatic residues in the pocket allow the charged and aromatic residues of the peptides to strongly interact with FPR1 by ionic-aromatic and stacking interactions, H-bonds, and salt bridges.Then, since in the case of C-terminal domain of FPR1 we did not find structures that can be used as templates, we modelled this domain using TM5 Fig. . This isTo characterize the interactions between FPR1 and fMLF, we first performed MD simulations on fMLF, linearly modeled as reported in the Methods. In Supplementary Fig. The best scored binding configuration between fMLF and FPR1 was selected on the basis of the number of interacting residues, H-bonds and salt bridges. In this complex, Phe3 is surrounded by: i) Phe81, Arg84 and Lys85 on TM2 that are reported as anchor region; ii) Trp91 on extracellular loop 1; iii) Val101 and Phe102 on TM3; iv) Asp284 on TM7. Hence, the interaction of Phe3 with FPR1 is based on three charged and four hydrophobic residues Fig. . On the 38. The analysis uncovered the occurrence of a network of MM H-bonds that starts from the H-bonds between the formylated Met1 of fMLF and Ser287, and proceeds from Ser287 to the C-terminal region by crossing TM7  the binding pocket of FPR1 comprises a localized region between the transmembrane helices TM2, TM3, TM5, TM6 and TM7; and iii) fMLF-engaged FPR1 displays an activation signature which reaches the FPR1 C-terminal loop by crossing helix 7.To understand how fMLF is able to activate FPR1 and to function as an agonist, we created an interaction network between amino acids, based on the interaction structure of residues Fig. . This apTM7 Fig. . This ne41. In our network, Ser328 H-bonds with Leu324 and Ser332, and in turn it forms H-bond with Gln330, whereas Ser338 forms two H-bonds with Thr336 and Leu340. These findings confirm the central role of these three residues in the H-bond network involved in signaling, and fit well with the ability of fMLF to trigger FPR1 internalization and signaling upon binding to FPR115.It is documented that the C-terminal region is crucial for internalization, desensitization, and arrestin2 binding and that Ser328, Ser332, and Ser338 located in this region regulate these processesTo characterize the interactions between FPR1 and SRSRY, MD simulations were performed on the SRSRY peptide, linearly modeled as reported in the Methods. RMSD plot for this peptide shows fluctuations with the highest RMSD values equal to 0.35 nm after 20 ns and 60 ns , the best scored binding configuration between the peptide and FPR1 was selected on the basis of the number of interacting residues, H-bonds and salt bridges. In this complex Fig. , Tyr5 isComparing the two complexes fMLF/FPR1 and SRSRY(turn)/FPR1, we found that in the SRSRY(turn)/FPR1 complex: i) there is also Phe291 of FPR1 in the anchor region; ii) the interaction between SRSRY(turn) and FPR1 in the anchor region is stabilized not only by the H-bond between Tyr5 and Lys85, but also by an additional H-bond between Tyr5 and Asp284. These observations suggest that the binding sites of fMLF and SRSRY (turn) are very similar/adjacent, even if not perfectly identical.The analysis based on interaction network of the complex between SRSRY(turn) and FPR1 highlighted that, as for the fMLF-FPR1 complex, there is a network of MM H-bonds that starts from the H-bonds between Arg4 of SRSRY and Ser287 and proceeds from Ser287 to the C-terminal region by crossing TM7 Fig. . This suThen, we modeled the complex between the extended conformation of SRSRY, SRSRY(extended), and FPR1. In this complex, Tyr5 is surrounded by Phe81, Arg84 and Lys85 on TM2, Trp91 and Phe93 on extracellular loop1, Asp284 and Phe291 on TM7, whereas Ser3 is in contact with Asp106 on TM3, Arg201 and Arg205 on TM5, Trp254 and Trp257 on TM6 and Ser287 on TM7 Fig. . In the Accordingly, we found that, like fMLF, 10 nM SRSRY promotes directional migration of only RBL-2H3 cells stably transfected with human FPR1 (RBL-2H3/ETFR), the extent of fMLF and SRSRY cell motility being quite comparable Fig. . Interes33. Now, we present evidence that RI-3 failed to trigger migration of both FPR1-lacking and FPR1-expressing RBL-2H3 cells  which does not exert any chemotactic activity and inhibits both fMLF- and SRSRY-directed cell migration to a similar extent as compared to unlabeled RI-3 Figs  and 7a. lls Fig. . Fluoromlls Fig. . In contlls Fig. . FITC-RII-3 Fig. .Figure 842, ensures its capability to prevent engagement, internalization and signaling of FPR1 by either fMLF and SRSRY. Binding experiments carried out on HEK-293 and HEK-293/uPAR cells that are documented to express FPR143, allowed us to ascertain that, when expressed on cell surface, uPAR does not affect the binding of FITC-RI-3 to FPR1. Moreover, unlike FITC-fMLF, FITC-RI-3 does not trigger FPR1 internalization in HEK-293/uPAR cells  RI-3 reached convergence after 20 ns with a mean value of RMSD around 0.3 nm; ii) RG plot for this peptide tended to decrease; iii) RI-3 formed a turn structure during the great part of the MD simulation with the formation of one MM H-bond between the CO group of D-Tyr1 and NH group of D-Arg4, specific of turn conformationThe best scored binding configuration between RI-3 and FPR1 was selected on the basis of the number of interacting residues, H-bonds and salt bridges. In this complex, D-Tyr1 is surrounded by Phe81, Arg84 and Lys85 on TM2, Trp91 on the extracellular loop1, Val101 and Phe102 on TM3 and Asp284 and Phe291 on TM7 whereas the residues Aib3 and D-Arg4 are surrounded by Asp106 on TM3, Arg201 and Arg205 on TM5 and Trp254 and Tyr257 on TM6 Fig. . In detaAltogether, our biological findings fit well with MD simulations of the FPR1/RI-3 complex. RI-3 competes with fMLF and SRSRY for binding to FPR1 and inhibits both fMLF- and SRSRY-directed cell migration by preventing agonist-triggered FPR1 internalization. Accordingly, RI-3 binds strongly FPR1 in the anchor region and shares the same binding site of fMLF and SRSRY on FPR1, located between TM2, TM3, TM5, TM6 and TM7 helices, but fails to engage the residues in the activation region. Therefore, in the absence of any stimulus, RI-3 does not promote neither cell migration, nor FPR1 internalization.Cell migration offers rich targets for intervention since its control may improve pathologic conditions sustained by an altered cell motility, including chronic inflammation and neoplastic diseases.32. More recently, in order to overcome the liabilities of these inhibitors due to their peptidic nature, we developed new retro-inverso peptides. Among these, the retro-inverso peptide RI-3 was identified as the best inhibitor of cell migration. RI-3 adopts a turn structure very similar to that adopted by previous characterized uPAR-FPR1 antagonists, is stable in human serum, prevents the uPAR/FPR1 interaction and potently inhibits migration and invasion of human sarcoma and melanoma cells34. Furthermore, RI-3 does not change the phosphorylation of protein kinases in the absence of any stimulus, but prevents fMLF-triggered activation of p38 MAPK and PI3K/AKT signaling cascades, without affecting cell proliferation34.In the past years, we developed uPAR-derived synthetic peptides that inhibit the uPAR/FPR1 interaction and reduce to the basal level the motility of cancer cellsTo understand the structural basis of RI-3 inhibitory effect on cell migration, in the absence of published crystallographic structures of FPR1, we decided to model the structure of this receptor and of the three FPR1/fMLF, FPR1/SRSRY and FPR1/RI-3 complexes, focusing the attention to the binding pocket of FPR1 and the interaction network of amino acids that transmits the binding information throughout the complex to the FPR1 C-terminal loop, triggering the signaling cascade.45, we identified a well-defined binding pocket of FPR1 which can accommodate only one peptide at a time, and is characterized by positively charged residues located in TM2 and TM5, by negatively charged residues on TM3, and by aromatic residues on TM6 and TM7. The peptides interact strongly with the charged and aromatic residues in the pocket by H-bonds, salt bridges and ionic-aromatic and stacking interactions. Comparison of the FPR1/fMLF and FPR1/SRSRY complexes uncovered that fMLF and SRSRY share a similar/adjacent binding site on FPR1, located between TM2, TM3, TM5, TM6 and TM7, and that the network of interactions through which SRSRY triggers signaling is the same found in the fMLF/FPR1 complex. fMLF recognizes FPR1 in a preferentially extended conformation as already reported by He and coworkers40, whereas SRSRY is more flexible and recognizes FPR1 either in extended or in compact conformation. However, in the extended conformation the binding of SRSRY to FPR1 is less stabilized than in the turn conformation. These differences may be explained considering that Ser90 is positioned in a critical hinge which influences the conformation of nearest residues16. Considering that uPAR may reversibly acquire distinct conformational states transitioning from an open and inactive to a closed and active conformation, and that uPA engagement shifts the inactive structure of uPAR to a closed, active conformation11, it is conceivable to hypothesize that FPR1/SRSRY(turn) and FPR1/SRSRY(extended) complexes could reflect intermediate stages of transition between the open/inactive to closed/active conformation of uPAR. Thus, uPA, beside focusing of its proteolytic activity on cell surface, could ultimately regulate the formation of uPAR/FPR1 complexes.In keeping with the notion that the ligand binding pocket of FPR1 consists of several key residues located in different transmembrane helices−11 M, and 3 × 10−8 M, respectively15). The relevance of this observation is not obvious considering that uPAR overexpression has been found in tumor tissues of different origin and soluble forms of uPAR containing the uPAR84-95 sequence shed from tumor cells and enrich cancer microenvironment with a strong pro-chemotactic factor46. Furthermore, the finding that, unlike fMLF, RI-3 does not trigger FPR1 internalization, either in the presence, or in the absence of uPAR, suggests that FPR1 is necessary to promote cell migration, and that the potency of chemotactic uPAR84-95 sequence is mainly mediated by FPR1.We also present evidence that RI-3 inhibits cell migration of human cells expressing a considerable amount of uPAR on the cell surface, in spite of the fact that RI-3 competes with SRSRY for binding to FPR1. This apparent contradiction may be reconciled considering that FPR1 displays an about 1000-fold higher binding affinity to RI-3 than to SRSRY  shares the same binding site of fMLF and SRSRY on FPR1 located between TM2, TM3, TM5, TM6 and TM7 helices; ii) strongly binds FPR1 in the anchor region, as indicated by three stacking and one π-cation interactions in which D-Tyr1 is involved, and iii) does not interact with the activation region of FPR1, thus being unable to activate the signaling cascade. These observations fit well with the finding that RI-3 binds to FPR1 with high affinity (apparent Kd:1 × 10−11 M) without triggering its internalization, nor cell motility. In agreement with docking studies, RI-3 can be regarded as a true FPR1 antagonist, since it does not elicit by itself the migration of either FPR1-lacking or FPR1-expressing cells, but reduces to basal level the migration of FPR1-expressing cells toward both SRSRY and fMLF, the latter with an IC50 of 1 × 10−13 M.Previous work from this laboratory has shown that RI-3 forms a turn structure during the great part of the MD simulation, with the formation of one MM H-bond between the CO group of D-Tyr1 and NH group of D-Arg4, specific of a turn conformationThe addition of an ε-aminocaproic acid spacer to the C-terminal amidated group of RI-3 in FITC-RI-3 did not modify significantly its binding properties. This information, together with the docking analysis of the FPR1/RI-3 complex may provide sufficient information for the design of peptide derivatives labeled with therapeutic radioisotopes, to elicit additional cytotoxic activity against cancer cells. Furthermore, the new compounds could be radiolabeled for micro-PET imaging in order to monitor their distribution and pharmacokinetics simultaneously in animal models.47. In this context, the selective inhibition of the uPAR/FPR1 interaction could facilitate the development of selective inhibitors of the processes sustained by a chronic excess of cell migration, such as inflammatory diseases, tumor spread and metastases, without affecting other functions regulated by FPR1.During the last twenty years, a very large number of natural and synthetic compounds that interact and/or interfere with FPR1-dependent pathways and several agonists and antagonists of FPR1-dependent functions have been describedab-initio. In detail, we modeled the N-terminal domain (region 1-27)and the trans-membrane region (region 28-305), composed by seven helices and by three extracellular and three cytoplasmic loops, by comparative modeling using as template the structure of type-1 angiotensin II receptor 48 and MODELLER program50. The C-terminal region (region 306-350)was modeled by Quark Server37. The best 3D models were selected on the basis of the energetic and stereo-chemical quality using ProSA program51 and Ramachandran plot52. Then, we have modeled the complete structure of FPR1 using as template the two structures obtained for N-terminal domain and the trans-membrane region and for C-terminal domain by MODELLER program50 as already reported in our recent paper38. FPR1 model has been deposited on Model Archive database and is accessible by the following link: 10.5452/ma-thpfw.Human FPR1 (Uniprot code: P21462) was modelled using an integrated modeling approach of comparative modelling and folding 53. Each peptide was inserted in a cubic box that contains water molecules and subjected to energy minimization and position restraints cycles. Then, MD simulations were performed for 100 ns at room temperature (300 K) by adding two chloride ions in the case of SRSRY to neutralize the net electrostatic charge of the system, as already published in the paper in which MD simulations on RI-3 are reported33. The trajectories were analyzed in terms of root mean squared deviation (RMSD), gyration radius fluctuations, root mean squared fluctuation (RMSF), secondary structure evolution and number of H-bonds using GROMACS routine.SRSRY and fMLF peptides were built by Builder module in Insight II and subjected to molecular dynamics (MD) simulations to study their energetic stability using GROMACS program and OPLS-AA force field54. In detail, we used our complete obtained model for FPR1 and the final conformations obtained after MD simulations for SRSRY, fMLF and RI-3 peptides. The selection of the best complexes was made by visual inspection, discarding models in which the peptides were not located in the binding groove, as reported in literature56, and selecting the complexes with the highest number of H-bonds and salt bridges by HBPLUS57 and ESBRI58 programs and the highest number of amino acids by LigPlot59.The complexes between FPR1 and three peptides  were modeled by molecular docking studies using Patchdock algorithm based on three major stages: molecular shape representation, surface patch matching and filtering/scoring60, HBPLUS57 and COCOMAPS61 tools were used to evaluate H-bonds, π-cations, π-stacking and IAC, respectively, that were inserted as interactions between the residues in the networks of FPR1-peptide complexes.For each FPR1-peptide complex, we analyzed the residue interaction network in which the amino acids are considered as nodes whereas the interactions between the residues as edges. In detail, Protein Interactions Calculator (PIC)2 (RI-3) peptides were custom-synthesized by JPT Peptide Technologies, Germany. The fluorescein-conjugate Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-Ahx-Lys (Nɛ-FITC)-NH2 (FITC-RI-3) peptide was custom-synthesized by MicroGem, Italy. These peptides were synthesized on solid-phase with Fmoc/t-Bu chemistry, purified by reversed-phase HPLC using water/acetonitrile gradients, and characterized by UPLC-MS. N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF) and the fluorescein-conjugated hexapeptide formyl-Nle-Leu-Phe-Nle-Tyr-Lys (FITC-fMLF) peptides were purchased by Invitrogen.The SRSRY, ARARY and Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-NH62 were kindly provided by F. Blasi . Human embryonic kidney HEK-293 cells which do not express uPAR and HEK-293 cells stably transfected with expression vector pcDNA3-uPAR (HEK-293/uPAR) have been previously described16. All cell lines were grown adherent in Dulbecco Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum supplemented with 100 IU/mL penicillin and 50 μg/mL streptomycin and maintained in an atmosphere of humidified air with 5% CO2 at 37 °C.Rat basophilic leukemia RBL-2H3 cells which do not express FPR1 and RBL-2H3/ETFR cells stably transfected with human FPR1 cDNA34. Briefly, cell suspension (1 × 105 viable cells/mL serum free medium) was seeded in each upper chamber. A subset of experiments were performed using cells desensitized with 100 nM fMLF or 100 nM SRSRY for 30 min at 37 °C in humidified air with 5% CO2 as described14. Lower chambers were filled with serum free DMEM containing diluent or the indicated peptides. The two compartments were separated by 8 μm pore size polycarbonate filters (Neuroprobe). Cells were allowed to migrate for 4 hr at 37 °C, 5% CO2. At the end of each assay, cells on the lower filter surface were fixed with ethanol, stained with haematoxylin and 10 random fields/filter were counted at 200x magnification. Each experiment was performed three times in triplicate.Cell migration in Boyden chambers was carried out as described6 cells/sample) were pre-incubated with diluents (CTRL), 1 μM fMLF, 1 μM SRSRY, 1 μM ARARY, 1 μM RI-3 or increasing concentration of RI-3 for 30 min at 4 °C, extensively rinsed with phosphate buffer saline (PBS), exposed to 10 nM FITC-RI-3 diluted in binding buffer (PBS containing 25 mM Hepes), for additional 45 min at 4 °C and again rinsed with PBS. Then, cells were visualized with the fluorescent Axiovert 200 microscope (Carl Zeiss). Quantification of cell-associated fluorescence was assessed by reading cells with a fluorescence plate reader Victor 3 (Perkin Elmer) using 485 nm excitation and 535 nm emission filters as already reported34. The experiments were performed three times in triplicate.Cells  Mowiol, visualized with a Zeiss 510META-LSM microscope (Carl Zeiss), and z-series were collected at 0.19 µm intervals as reportedData from cell migration and binding assays were analyzed for significance using the Student’s t-test.All experimental protocols were performed in accordance with guidelines of the Istituto Nazionale Tumori IRCCS “Fondazione G. Pascale” .Supplementary Information


These findings suggest that a given host’s susceptibility to HAM/TSP is deeply connected with both differences in genetically determined components of the host immune response and HTLV-1 subgroup. Therefore, it is crucial for ongoing work to focus on developing novel treatments and preventative approaches for HAM/TSP. In this review, based on an overview of the topic and our latest research findings, the role of the HTLV-1 subgroup on the effects of virus–host interactions in the pathogenesis of HAM/TSP is discussed.Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disorder presenting with spastic paraparesis, sphincter dysfunction, and mild sensory disturbance in the lower extremities, which develops in a small minority of HTLV-1-infected individuals. HTLV-1-specific T cells are efficiently activated through dedicated human leukocyte antigen-mediated mechanisms, a process considered deeply involved with its pathogenesis. It has been reported that the lifetime risk of developing HAM/TSP differs between ethnic groups, and there is an association between HTLV-1  Human T-cell leukemia virus type 1 (HTLV-1), the first human retrovirus to be discovered, is etiologically associated with adult T-cell leukemia (ATL)  and HTLVHTLV-1-associated myelopathy/tropical spastic paraparesis is a rare chronic progressive neuroinflammatory disease of the central nervous system mainly involve the thoracic spinal cord, therefore characterized by spastic paraparesis, sphincter dysfunction, and mild sensory disturbance in the lower extremities . Based oin vitro culture of peripheral blood mononuclear cells (PBMCs) from HTLV-1-infected individuals exhibit “spontaneous lymphocyte proliferation” (SLP) in the absence of exogenous stimuli such as antigens or mitogens. In patients with HAM/TSP, the levels of SLP reflect the severity of the disease (∗0101-restricted dominant epitope on HTLV-1 envelope (Env) glycoprotein gp21 (in vivo because HTLV-1-infected cells in vivo harbor one copy of the integrated HTLV-1 provirus per cell (in vivo) as a risk factor for HAM/TSP  are most commonly found on HTLV-1 regulatory factor Tax; HLA-A∗02-restricted epitope on Tax, spanning amino acids 11–19, is known to provoke a particularly intense immune response , southwest Japan, Australia , the Caribbean, and South America . In thesectively . As show strains , which r∗02 provides protection against tax subgroup-B, but not against tax subgroup-A in both the Japanese . Thus, it is noteworthy that Jamaican individuals have a higher incidence of “cosmopolitan subtype 1a subgroup A” infection  compared with Japanese individuals with a higher incidence of the “cosmopolitan subtype 1a subgroup B” infection . Namely,Japanese  and IranJapanese  populati∗02 and the risk of HAM/TSP, possessed 10 different nucleotides in the tax region compared with Japanese prototypic strain ATK-1 (= tax subgroup B)  is susceptible to HAM/TSP compared with the Japanese subgroup (= tax subgroup B). These findings suggested that a high-risk subgroup of HTLV-1 explains the observed differences in HTLV-1 PVL and higher risk for HAM/TSP.Interestingly, HTLV-1 in Iranian individuals, which showed no association between HLA-Agroup B) . Among tbgroup A . Because strains , it is ptax associated with HAM/TSP morbidity  and Japanese (B) subgroups within cosmopolitan subtype 1a, a taxonomy defined based on LTR sequence similarity  based on reporter gene assays using three different reporters, i.e., the HTLV-1-3′-LTR-promoter luciferase reporter, human Foxp3 promoter luciferase reporter, and human TGF-β-responsive concatemer-containing luciferase reporter  potently activated the expression of their target genes, and Tax-A and -B elicited the expression of different combinations of both already-reported and unreported target genes (More recently, we further studied the function of each subgroup-specific Tax using microarray analysis, reporter gene assays, and evaluation of viral–host protein–protein interactions . In thiset genes . Furtheret genes , was moret genes . Meanwhiet genes .tax subgroups still remain to be fully clarified. Moreover, to date, no research has observed subgroup-based differences in the ability of Tax to regulate transcription via independent mechanisms of direct transcriptional regulation nor compared HTLV-1 immunogenicity between subgroups A and B. Future research in these directions is eagerly anticipated to identify attractive targets for novel therapeutics for HAM/TSP.As discussed above, each subgroup-specific Tax or HBZ have a unique expression signature of both viral and host genes, suggesting that differential expression of pathogenesis-related genes by subgroup-specific viral regulatory proteins may be associated with the onset of HAM/TSP. However, the associations between HAM/TSP susceptibility and MS wrote this review manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lymph nodes (LNs) are crucial for the orchestration of immune responses. LN reactions depend on interactions between incoming and local immune cells, and stromal cells. To mediate these cellular interactions an organized vascular network within the LN exists. In general, the LN vasculature can be divided into two components: blood vessels, which include the specialized high endothelial venules that recruit lymphocytes from the bloodstream, and lymphatic vessels. Signaling via TNF receptor (R) superfamily (SF) members has been implicated as crucial for the development and function of LNs and the LN vasculature. In recent years the role of cell-specific signaling of TNFRSF members in different endothelial cell (EC) subsets and their roles in development and maintenance of lymphoid organs has been elucidated. Here, we discuss recent insights into EC-specific TNFRSF member signaling and highlight its importance in different EC subsets in LN organogenesis and function during health, and in lymphocyte activation and tertiary lymphoid structure formation during inflammation. Lymph nodes (LNs) are positioned at strategic sites throughout the body where they are essential for initiating and shaping immune responses. Via the vascular system soluble factors and cells are transported from peripheral tissues into the LNs, which is crucial for the initiation of (adaptive) immune responses , 2. LympGenerally, LNs can be divided into three areas; the cortex containing B cell follicles, the paracortex consisting predominantly of T cells, and the medulla representing the primary maturation site of antibody producing plasmablasts , 2. ThisArterial blood that enters the LN flows into capillaries which proceed into the network of HEVs. High endothelial venules located within the T cell areas are the sites were naïve lymphocytes leave the bloodstream to enter the LN and interact with local cells. After passing through the HEVs venous blood exits the LN via the efferent blood vessels , 6. AffeAmong the signaling cascades that have been recognized as essential for LN development is signaling via the tumor necrosis factor receptor (TNFR) superfamily (SF). It is well-established that signaling of TNFRSF members in many cell types is required for proper LN development and function, including LN EC subsets.Here, we present an overview of the EC-specific TNFRSF member signaling cascades that are important for LN organogenesis and development and maintenance of the different LN EC subsets. In addition, the importance of these cells and signaling pathways during inflammation are discussed, focusing on LN inflammatory reactions and development of tertiary lymphoid structures (TLSs). Lastly, we will discuss whether targeting of EC-specific TNFRSF member signaling may hold potential as a therapeutic target in the treatment of inflammatory diseases.+CD4++IL-7Rα+ lymphoid tissue inducer (LTi) cells are recruited into the LN anlagen (− pre-LTi cells egress from venous vessels at locations where there is low coverage of smooth muscle cells (SMCs) (+ LTi cells that are transported to and retained at the site of LN development by the LVs (Development of LNs starts around embryonic day (E) 9 at the same time that ECs start budding from the anterior cardinal vein and begin to express the lymphatic EC (LEC) marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Between E12.5-14.5, CD45 anlagen , 9 a pro anlagen . Recentls (SMCs) . These p the LVs .1β2  on LTi cells that engages the LTβ receptor  on the surrounding stromal cells  cells is necessary to further support retention of LTi cells at the LN anlage -1, vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1, and the chemokines C-X-C motif ligand (CXCL)13, C-C motif ligand (CCL) 19 and CCL21  signaling . This siRankl−/−  from tissues into the LNs . CharactAfferent LVs originating from the peripheral tissue branch into the SCSs located directly underneath the LN capsule, extend into the T and B cell areas, and exit as efferent vessels , 70. VialowVEGFR+ collecting LVs between E15.5-E16.5  B cells, suggesting concerted action of the LTβR and B cells in lymphangiogenesis and HEVs plasticity  . TLSs rerent LVs , 97. Emerent LVs –100. Therent LVs –101 and 1β2 or TNFα under the rat insulin promotor (RIP-LTα1β2 and RIP-TNFα transgenic (tg) mice, respectively) spontaneously develop infiltrates consisting of T and B cells in the pancreas   , 103. Itormation , 104–106+ HEC-like cells. During chronic inflammation the blood vasculature undergoes remodeling in order to recruit immune cells into the inflamed tissue. This remodeling involves upregulation of adhesion molecules and production of chemoattractants by BECs  immune cells into the tissue to mediate interaction with cognate antigens leading to immune cell activation and memory cell formation (+ BVs may act anti-tumoral via recruitment of naïve and memory T cells (In general, TLS associated PNAdormation , 110–112ormation , 114. Inormation , 96. In  T cells –117, but T cells .+ BVs in TLSs seems to be LTβR dependent. It has been shown that initiation of TLS-PNAd+ BVs occurs via the LTα-TNFRI-canonical NF-κB axis leading to MAdCAM-1+ BVs and that LTα1β2-LTβR-noncanonical NF-κB signaling generates PNAd+ BVs (+ BVs located in the synovial tissue of rheumatoid arthritis patients are NIK+ comparable to LN HEVs (+ BVs (+ BVs are formed in absence of real TLSs, PNAd+ BVs can also develop via LTα-TNFRI signaling, independent of LTβR signaling (+ BVs. However, the function of these vessels is shown to vary between pathologies, making it hard to predict the outcome of targeting HEVs during disease.Similar to LN HEVs, formation of PNAdNAd+ BVs , 98, 118 LN HEVs . In addis (+ BVs . Althougignaling . TogetheThe role of LVs in inflammation has long been recognized; i.e., LVs play an immunoregulatory role in inflammation via fluid drainage, scavenging of inflammatory chemokines and suppression of DC maturation , 122. Du+ Prox-1+, PDPN+CCL21+ vessels are found in TLSs of many pathologies, their exact role and function is not well-understood (Although LYVE-1derstood , 126. Thderstood . In contderstood . It has derstood .1β2 is not required (1β2 might even negatively regulate lymphangiogenesis since Ltb−/− mice exhibit increased lymphangiogenesis, which may be due to the fact that mice lacking LTα1β2 have more LTα available to form trimers that bind to TNFRI (In mice lacking LTβR, TNFRI or LTα, the development of LVs in the inflamed area is impaired, while the surrounding LVs remain largely unaffected , 129. ItThe role of RANK signaling in TLS associated LVs has not yet been fully addressed, but with the recent evidence demonstrating an important role for that RANK signaling in LECs in the development of mature functional LNs , it willIt is clear that signaling through TNFRSF members is crucial for development and functioning of LNs and TLSs, both in health and disease. Here, we emphasized the importance of EC-specific TNFRSF member signaling cascades in these processes.+ BVs.It is clear that TNFRSF signaling, mainly via the LTβR, in BECs is crucial for their differentiation into HECs, which subsequently regulate recruitment of lymphocytes into the LNs. In addition, the same TNFRSF-mediated mechanisms are involved in formation of TLS associated PNAdAlthough considerably less is known about TNFRSF member signaling in LVs, recent data does point toward an important role for these pathways in LEC function in LN development and function. A prominent role seems to be reserved for signaling via RANK, although LTβR signaling is also likely to be involved. Interestingly, different mechanisms may be involved in functioning of LECs in LNs and TLSs.The currently available studies aimed at unraveling the role of signaling pathways in LN EC subsets already prompt possible refinements of the existing models for LN development and the role of TNFRSF member signaling in ECs within this process, but there are still several outstanding questions. One of these questions is to what extent the timing of TNFRSF signaling is important, since it is not fully understood at what exact time point signaling events in specific EC subsets are crucial for the development of fully functional lymphoid organs. In this respect, the development of EC-specific conditional KO mice is very promising and aid tremendously in addressing this issue.+ BVs and other blood vessels in inflamed tissues, but not in healthy tissues (In line with this, it will also be interesting to see if interfering with specific signaling pathways in EC subsets holds therapeutic potential in treatment of chronic inflammatory diseases and other pathologies. Several clues point toward this, for example the finding that NIK can be detected in TLS associated PNAd tissues . Here, cIt is likely to assume that in the near future many outstanding questions will be answered. With the current possibilities in animal models and advanced techniques to compare patients with healthy individuals, it is only a matter of time until deeper insights into the cell specific signaling pathways in development and maintenance of LNs and TLSs are gained. More importantly, it will be of great interest to uncover how these pathways contribute to disease and whether they hold therapeutic potential.KJ: conceived the general idea, wrote the manuscript, and created the figures. JK and RM: provided expert opinion/knowledge input, and co-wrote the manuscript. ST: conceived the general idea, provided expert opinion/knowledge input, and co-wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ursolic acid (UA) is an antitumor component derived from Chinese herbal medicine; this study is to observe the effects of UA on epithelial-mesenchymal transition (EMT) in gastric cancer. In vitro experiments: 25μmol/L and 50μmol/L UA were applied to BGC-823, AGS, MGC-803, and HGC-27 cells; MTT staining, Transwell assay, and flow cytometry were used to assess cell proliferation, cell migration, and apoptosis, respectively. Western blot was performed to detect the expressions of N-Cadherin, Vimentin, Snail, Twist, Axl, p-Axl, IKK, p-IKK, NF-κB, and p-NF-κB. (2) In vivo experiments: Ten BALB/c-nu mice were used to establish gastric cancer xenograft model. Five were orally given UA for 4 weeks and five were given normal saline. Expressions of N-Cadherin and Snail were examined by immunohistochemical assay; expressions of N-Cadherin, Snail, Twist, Axl, p-Axl, IKK, and p-IKK were detected by Western blot. (1)α/β, and p-NF-κB in BGC-823 and MGC-803 cells. (4) UA distinctly decreased the expressions of N-Cadherin, Snail, p-Axl, and p-IKKα/β in gastric cancer xenograft model rats. (1) UA inhibited cell proliferation in BGC-823 and HGC-27 cells in dose-dependent manners. (2) UA inhibited cell migration in BGC-823, AGS, and MGC-803 cells while inducing apoptosis in BGC-823 cells. (3) UA significantly decreased the expressions of N-Cadherin, Vimentin, Snail, Twist p-Axl, p-IKKκB signaling pathway. UA can effectively inhibit the proliferation and migration and induce apoptosis of gastric cancer cells. The antitumor effect of UA is conducted by EMT inhibition, which may be associated with the regulation of Axl/NF- Gastric carcinoma (GC) is the fourth most common malignant tumor throughout world, and it is the second leading cause of cancer mortality . In ChinThe epithelial-mesenchymal transition (EMT) is a process through which epithelial cells are converted into mesenchymal cells; it is characterized as the loss of cell-cell adhesion and cell polarity, the acquisition of migratory and invasive properties, and the improvement of resistance to apoptosis and degradation of extracellular matrix . TherefoUrsolic acid (UA) is an antitumor component derived from Chinese herbal medicine. The structural formula is shown in  in vivo and in vitro experiments to observe the effects of UA on EMT in GC. In in vitro experiment, BGC-823, AGS, MGC-803, NCI-N87, and HGC-27 cells were used; MGC-803 represents high differentiation stage of GC, BGC–823 and AGS represent poorly differentiated GC, HGC-27 represents undifferentiated GC, and NCI-N87 represents fore-GC. In in vivo experiment, gastric cancer xenograft mice model was used, since it well simulates the pathogenesis and pathological change of human gastric cancer.In this study, we conducted bothGastric cancer cell lines BGC-823, HGC-27, AGS, MGC-803, and NCI-N87 were from Central Research Center, Zhejiang Province Hospital of Traditional Chinese Medicine.2 cell culture flask with a density of 2×106/mL. Cells were maintained in RPMI-1640 medium with 10% calf serum and grew at 37°C in a CO2 incubator. Cells in logarithmic growth phase were collected for following experiments.After cell resuscitation, cells were inoculated in a 25cmμL of BGC-823 and HGC-27 (3×105/mL) were added to each well in the 96-well plate. UA was added to the final concentration of 6.25μmol/L, 12.5μmol/L, 25μmol/L, 50μmol/L, and 100μmol/L, respectively. Dimethyl sulfoxide and culture medium were added as control. 10μL Cell Counting Kit-8 was added, plates were lucifuge incubated in 37°C for 1h, and the absorbance values were examined. The inhibiting proliferation rates were calculated using the following equation: Inhibiting proliferation rate (%) = / a value of control group×100%.250μL of AGS, HGC-27, MGC-803, and NCI-N87 were inoculated in the upper chambers, and the cell density was adjusted to 1.5×104/mL and 2×104/mL, respectively. 500μL of culture medium with 10% calf serum was added in the lower chambers; the chambers were incubated at 37°C, 5% CO2 for 24h, then rinsed with phosphate buffer saline, fixed with 4% formalin for 20min, and crystal violet stained for 20min. Cotton swabs were used to remove the cells that are unable to penetrate the membrane surface. Photograph under 200× magnification and count the number of migrated cells.100μL of UA was added to AGS, MGC-803, and BGC-823 cells with the final concentration of 25μmol/L and 50μmol/L, respectively; after incubation for 24h, the Transwell assay was performed again according to the above steps.According to the above experimental results, 105/mL) were treated with UA at concentration of 50μmol/L for 24h. Cells were collected after centrifugation, 300μL 1×Binding Buffer was added to resuspend the cells, 5μL Annexin V-FITC Early Apoptosis Detection Kit (bioSmile), and 5μL propidine iodide was added and lucifuge incubated for 15min and 5min under room temperature, respectively. Then 200μL 1×Binding Buffer was replenished; flow cytometry was used for apoptosis analysis.BGC-823, MGC-803, and AGS cells (1.5×105/mL) were treated with 25μmol/L and 50μmol/L UA; expressions of N-Cadherin, Vimentin, Snail, Twist, Axl, p-Axl, IKKα/β, p-IKKα/β, NF-κB, and p-NF-κB were examined by Western blot. Cells were treated with RIPA Lysis Buffer . Concentrations of proteins were determined by bicinchoninic acid assay. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and incubated with proper primary and secondary antibodies. Then the proteins were transferred to polyvinylidene fluoride membrane, and the integrated option density was detected by far infrared scanner  and Image J software. The relative expressions of proteins were calculated by using β-actin as an internal control.BGC-823, MGC-803, and AGS cells .7/mL of BGC-823 cell suspension was injected into the axillary skin of 4 BALB/c-nu mice; condition was observed every 3 days. After 2 weeks, mice were sacrificed by carbon dioxide euthanasia when the tumor grew to a diameter of 1cm. The underarm skin was routinely disinfected; the tumor tissues were peeled and cut into small pieces about 1mm3.Establishment of subcutaneous xenograft tumors: 5×10Establishment of GC xenograft model: Ten nude mice were anaesthetized by intraperitoneal injection of pentobarbital sodium peritoneal (50mg/kg); a 0.2cm long incision was cut in the right anterior armpit. The tumor was then implanted into the incision and a drop of OB glue was put to bind the incision. The condition of mice was observed every day to determine the tumorigenesis. At 72h after successful modeling, mice were randomly divided into the model group and UA group; each has 5 mice. UA group was orally given UA 50mg/kg/d, and corresponding saline was given to the model group for four weeks. Under anesthesia by pentobarbital sodium, the tumor tissues were removed, and mice were sacrificed by carbon dioxide euthanasia.Expressions of N-Cadherin and Snail in mouse GC xenograft model were examined by immunohistochemistry staining. Sections were routinely dewaxed and hydrated. After the heat-mediated antigen retrieval, the sections were blocked with 10% goat serum at 37°C for 1 h. Then the sections were incubated with primary antibody (1:800) at 4°C overnight. Polink-1 horse radish peroxidase-diaminobenzidine 3 detection system was used to incubate the sections at 37°C for 1 h, followed by diaminobenzidine 3 coloration and haematoxylin restaining. The integral optical density was analyzed by Image Pro Plus 6.0 software.α/β, and p-IKKα/β in mouse GC xenograft model were determined by Western blot. Tumor tissues were homogenized, and the following operations were as same as above.Expressions of N-Cadherin, Snail, Axl, p-Axl, IKKStatistical analysis was performed by using SPSS19.0 software. Data were presented as mean ± SD. One-way ANOVA was used for comparison between groups. Least-significant difference test was applied when the variance was equal; otherwise Tamhane's T2 test was performed. A value of P < 0.05 was considered to be statistically significant.μmol/L) inhibited cell proliferation in dose-dependent manners both in BGC-823 and in HGC-27 cells.Proliferation of BGC-823 and HGC-27 cells was examined to assess the inhibitory effect on cell growth of UA. As shown in μmol/L UA was added, the apoptosis rate increased to 29.07%, which indicates that UA can significantly induce apoptosis in BGC-823 cells. UA damaged MGC-803 and AGS cells too much to obtain corresponding result.Apoptosis in BGC-82, MGC-803, and AGS cells was assessed by flow cytometry. As shown in 4 cells/well, while HGC-27 and NCI-N87 cells showed no distinct migration even at the density of 2×104 cells/well. Our previous study showed that BGC-823 cells migrated significantly under the condition of 5×104 cells/well. Therefore, AGS, MGC-803, and BGC-823 cells were selected for further experiments.The cell migration capacity was analyzed by Transwell assay. As shown in μmol/L) significantly inhibited BGC-823 cell migration in dose-dependent manners. 25μmol/L UA had obvious inhibitory effect on the migration of AGS and MGC-803 cells, but 50μmol/L UA damaged the two groups of cells, leading to the inability of subsequent experiments.As shown in μmol/L and 50μmol/L UA decreased the levels of N-Cadherin, Snail, Vimentin, and Twist in BGC-823 cells. 25μmol/L UA decreased the levels of N-Cadherin, Vimentin, and Twist in MGC-803 cells. 50μmol/L UA damaged the cells too much to obtain corresponding result in MGC-803 cells.The expressions of EMT related proteins were examined by Western blot. As shown in κB pathway were examined by Western blot. As shown in κB, and p-IKKα/β, while increasing the levels of Axl, NF-κB, and IKKα/β, and the values of p-Axl/Axl, p-IKK/IKK, and p-NF-κB/NF-κB were decreased. These results indicate that phosphorylations of Axl, IKK, and NF-κB have been inhibited by UA in BGC-823 cells.To investigate the mechanism on how UA regulates EMT, the expressions of key proteins in Axl/NF-Expressions of N-Cadherin and Snail in mouse GC xenograft model were examined by immunohistochemical analysis. As shown in κB pathway were examined by Western blot in mouse GC xenograft model. As shown in α/β, and p-IKKα/β, and the values of p-Axl/Axl and p-IKK/IKK significantly decreased (P<0.05). These results indicate that the phosphorylations of Axl and IKK have been inhibited by UA.The key proteins in Axl/NF- Oldenlandia diffusa and Radix actinidiae. It has the effects of anti-inflammatory, antioxidant, and antitumor [UA is a pentacyclic triterpene compound and exists in medicinal herbs such asntitumor . Studiesntitumor , inductintitumor , and prentitumor . In thisntitumor , 10.μmol/L UA decreased the levels of N-Cadherin, Vimentin, Snail, and Twist in BGC-823 and MGC-803 cells. UA significantly reduced N-Cadherin and Snail expressions in mouse GC xenograft model. The results verified the inhibition effects of UA on EMT in GC from both in vivo and in vitro.Metastasis is the leading cause of cancer deaths. Attenuation of intercellular adhesion and enhancement of cellular activity are the basis of tumor invasion and metastasis . EMT plaκB has the effect of promoting EMT [κB pathway had decreased [κB could block EMT and affect epithelial plasticity and metastasis of breast cancer; on the contrary, activation of NF-κB promotes EMT in breast cancer cells [κB integrates with its inhibitor IκB-α; under the activation of kinase IKKα, IKKβ, and IKKγ, IκB-α was phosphorylated and degraded, which expose the nuclear localization sequence of NF-κB and promote the activity of EMT. Shishodia et al. found that UA can inhibit IKKα and p65 phosphorylation, leading to the suppression of NF-κB activation induced by various carcinogens [κB pathway. Results show that 25μmol/L UA decreased the levels of p-Axl, p-IKKα/β, and p-NF-κB in BGC-823 cells, and it significantly decreased the p-Axl and p-IKK in mouse GC xenograft model. The results indicate that the inhibitory effects of UA on EMT may be associated with the regulation of Axl/NF-κB signaling pathway.EMT is regulated by many signaling pathways. Axl is a member of receptor tyrosine kinase family and involves in the regulation of cell adhesion, proliferation, and cell transformation . The oveting EMT . Kiefel ecreased . Huber eer cells . In the cinogens . To furtμmol/L UA damaged MGC-803 and AGS cells too much to proceed with subsequent experiments; further exploration of effective dose of UA is necessary.In this study, 50κB signaling pathway.UA can effectively inhibit the proliferation and migration and induce apoptosis of gastric cancer cells. The antitumor effect of UA is conducted by EMT inhibition, which may be associated with the regulation of Axl/NF-