Project description:EMILIN1 promotes ?4?1 integrin-dependent cell adhesion and migration and reduces pro-transforming growth factor-? processing. A knockout mouse model was used to unravel EMILIN1 functions in skin where the protein was abundantly expressed in the dermal stroma and where EMILIN1-positive fibrils reached the basal keratinocyte layer. Loss of EMILIN1 caused dermal and epidermal hyperproliferation and accelerated wound closure. We identified the direct engagement of EMILIN1 to ?4?1 and ?9?1 integrins as the mechanism underlying the homeostatic role exerted by EMILIN1. The lack of EMILIN1-?4/?9 integrin interaction was accompanied by activation of PI3K/Akt and Erk1/2 pathways as a result of the reduction of PTEN. The down-regulation of PTEN empowered Erk1/2 phosphorylation that in turn inhibited Smad2 signaling by phosphorylation of residues Ser245/250/255. These results highlight the important regulatory role of an extracellular matrix component in skin proliferation. In addition, EMILIN1 is identified as a novel ligand for keratinocyte ?9?1 integrin, suggesting prospective roles for this receptor-ligand pair in skin homeostasis.
Project description:The human S100A4 protein binds calcium, resulting in a change in its conformation to promote the interaction with its target protein. Human epidermal growth factor (EGF) is the target protein of S100A4 and a critical ligand of the receptor EGFR. The EGF/EGFR system promotes cell survival, differentiation, and growth by activating several signaling pathways. Amlexanox is an anti-inflammatory and anti-allergic drug that is used to treat recurrent aphthous ulcers. In the present study, we determined that amlexanox interacts with S100A4 using heteronuclear single quantum correlation titration. We elucidated the interactions of S100A4 with EGF and amlexanox using fluorescence and nuclear magnetic resonance spectroscopy. We generated two binary models (for the S100A4-EGF and S100A4-amlexanox complexes) and observed that amlexanox and EGF share a similar binding region in mS100A4. We also used a WST-1 assay to investigate the bioactivity of S100A4, EGF, and amlexanox, and found that amlexanox blocks the binding between S100A4 and EGF, and is therefore useful for the development of new anti-proliferation drugs.
Project description:Optogenetic approaches have gathered momentum in precisely modulating and interrogating cellular signalling and gene expression. The use of optogenetics on the outer cell surface to interrogate how cells receive stimuli from their environment, however, has so far not reached its full potential. Here we demonstrate the development of an optogenetically regulated membrane receptor-ligand pair exemplified by the optically responsive interaction of an integrin receptor with the extracellular matrix. The system is based on an integrin engineered with a phytochrome-interacting factor domain (OptoIntegrin) and a red light-switchable phytochrome B-functionalized matrix (OptoMatrix). This optogenetic receptor-ligand pair enables light-inducible and -reversible cell-matrix interaction, as well as the controlled activation of downstream mechanosensory signalling pathways. Pioneering the application of optogenetic switches in the extracellular environment of cells, this OptoMatrix-OptoIntegrin system may serve as a blueprint for rendering matrix-receptor interactions amendable to precise control with light.
Project description:We have previously shown that glioblastoma stem cells (GSCs) are enriched in the hypoxic tumor microenvironment, and that monocarboxylate transporter-4 (MCT4) is critical for mediating GSC signaling in hypoxia. Basigin is involved in many physiological functions during early stages of development and in cancer and is required for functional plasma membrane expression of MCT4. We sought to determine if disruption of the MCT-Basigin interaction may be achieved with a small molecule. Using a cell-based drug-screening assay, we identified Acriflavine (ACF), a small molecule that inhibits the binding between Basigin and MCT4. Surface plasmon resonance and cellular thermal-shift-assays confirmed ACF binding to basigin in vitro and in live glioblastoma cells, respectively. ACF significantly inhibited growth and self-renewal potential of several glioblastoma neurosphere lines in vitro, and this activity was further augmented by hypoxia. Finally, treatment of mice bearing GSC-derived xenografts resulted in significant inhibition of tumor progression in early and late-stage disease. ACF treatment inhibited intratumoral expression of VEGF and tumor vascularization. Our work serves as a proof-of-concept as it shows, for the first time, that disruption of MCT binding to their chaperon, Basigin, may be an effective approach to target GSC and to inhibit angiogenesis and tumor progression.
Project description:The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1DeltaG) are defective in regulating E2F target genes. Surprisingly, cell cycle regulation in Rb1DeltaG/DeltaG MEFs strongly resembles that of wild type. In a serum deprivation induced cell cycle exit, Rb1DeltaG/DeltaG MEFs display a similar magnitude of E2F target gene derepression as Rb1-/-, even though Rb1DeltaG/DeltaG cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1DeltaG/DeltaG MEFs is responsive to p16 expression, indicating that the DeltaG-pRB protein can be activated in G1 to arrest proliferation through non-E2F mechanisms. Some Rb1DeltaG/DeltaG mice die neonatally with a muscle degeneration phenotype, while the others live a normal lifespan with no evidence of spontaneous tumor formation. Histological analysis reveals discrete examples of hyperplasia in the mammary epithelium, but most tissues appear normal while being accompanied by derepression of pRB regulated E2F targets. This suggests that non-E2F, pRB dependent pathways may have a more relevant role in proliferative control than previously identified. Total RNA was extracted from littermate paired WT, DeltaG and null MEFs induced to enter quiencense by Serum deprivation. Expresssion levels were analyzed by Affymetrix GeneChip Mouse Gene 1.0ST Array. Relative expression was determined by BRB-Array Tools software to generate RMA values.
Project description:N-myc downstream-regulated gene 1 (NDRG1) is a multifunctional protein associated with carcinogenesis and tumor progression. The function of NDRG1 in hepatocellular carcinoma (HCC) cells remains controversial. The present study investigated the role of NDRG1 in HCC as well as its molecular mechanism using a range of techniques, including western blot analysis, cellular proliferation test, wound healing assay and Transwell assay. In HCC, the levels of NDRG1 expression were highest in the cytoplasm, followed by the membrane, and were lowest in the nucleus. NDRG1 was revealed to inhibit the proliferation and invasion of BEL7402 cells, which facilitated the hypothesis that NDRG1 expression levels may be lower in cell line with a high metastatic potential compared with those in cell lines with a low metastatic potential. However, the present study identified that NDRG1 expression was higher in detached BEL7402 cells and MHCC-97H cells compared with that in attached BEL7402 cells and MHCC-97L cells. Thus, this finding was contrary to what was expected, suggesting that NDRG1 overexpression in the HCC with a high metastatic potential may be the compensatory mechanism. The human HCC BEL7402 cell line demonstrated a significant increase in the capability of motility, invasion and cellular proliferation following NDRG1-short hairpin RNA transfection. Integrin β3 (ITGB3) protein expression was increased in NDRG1-downregulated BEL7402 cells and SMMC7721 cells compared with that in the control cells. The present study suggested that NDRG1 may be a potential anti-tumor target for the treatment of patients with HCC. A potential mechanism for these roles of NDRG1 is by regulating ITGB3 expression; however, this requires additional investigation.
Project description:MYC is a key player in tumor development, but unfortunately no specific MYC-targeting drugs are clinically available. MYC is strictly dependent on heterodimerization with MAX for transcription activation. Aiming at targeting this interaction, we identified MYCMI-6 in a cell-based protein interaction screen for small inhibitory molecules. MYCMI-6 exhibits strong selective inhibition of MYC:MAX interaction in cells and in vitro at single-digit micromolar concentrations, as validated by split Gaussia luciferase, in situ proximity ligation, microscale thermophoresis and surface plasmon resonance (SPR) assays. Further, MYCMI-6 blocks MYC-driven transcription and binds selectively to the MYC bHLHZip domain with a KD of 1.6?±?0.5??M as demonstrated by SPR. MYCMI-6 inhibits tumor cell growth in a MYC-dependent manner with IC50 concentrations as low as 0.5??M, while sparing normal cells. The response to MYCMI-6 correlates with MYC expression based on data from 60 human tumor cell lines and is abrogated by MYC depletion. Further, it inhibits MYC:MAX interaction, reduces proliferation and induces massive apoptosis in tumor tissue from a MYC-driven xenograft tumor model without severe side effects. Since MYCMI-6 does not affect MYC expression, it is a unique molecular tool to specifically target MYC:MAX pharmacologically and it has good potential for drug development.
Project description:Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against ?1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. ?1-integrin inhibiting antibodies bound to the vascular endothelium in vivo improved the integrity of endothelial cell-cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial ?1-integrin protected mice from LPS-induced vascular leakage. In endothelial monolayers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1? decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via ?1- and ?5-integrins and ANGPT2. Additionally, ?1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of ?5?1-integrin into tensin-1-positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, ?1-integrin promotes endothelial barrier disruption during inflammation, and targeting ?1-integrin signaling could serve as a novel means of blocking pathological vascular leak.
Project description:Acute myeloid leukemia (AML) with granulocytic sarcoma (GS) is characterized by poor prognosis; however, its underlying mechanism is unclear. Bone marrow samples from 64 AML patients (9 with GS and 55 without GS) together with AML cell lines PL21, THP1, HL60, Kasumi-1, and KG-1 were used to elucidate the pathology of AML with GS. RNA-Seq analyses were performed on samples from seven AML patients with or without GS. Gene set enrichment analyses revealed significantly upregulated candidates on the cell surface of the GS group. Expression of the adhesion integrin ?7 (ITGA7) was significantly higher in the GS group, as seen by RT-qPCR (p = 0.00188) and immunohistochemistry of bone marrow formalin-fixed, paraffin-embedded (FFPE) specimens. Flow cytometry revealed enhanced proliferation of PL21 and THP1 cells containing surface ITGA7 in the presence of laminin 211 and stimulated ERK phosphorylation; this effect was abrogated following ITGA7 knockdown or ERK inhibition. Overall, high ITGA7 expression was associated with poor patient survival (p = 0.0477). In summary, ITGA7 is highly expressed in AML with GS, and its ligand (laminin 211) stimulates cell proliferation through ERK signaling. This is the first study demonstrating the role of integrin ?7 and extracellular matrix interactions in AML cell proliferation and extramedullary disease development.
Project description:Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin ?v?3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3? activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.