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Experimental & Molecular Medicine Sep 2018Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer with poor prognosis. In the present study, we demonstrated that Src, a non-receptor tyrosine...
Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer with poor prognosis. In the present study, we demonstrated that Src, a non-receptor tyrosine kinase, might provide an effective therapeutic strategy to overcome TNBC invasion and metastasis, which are mediated via the synergistic action of the lysosomal enzyme cathepsin S (CTSS) and gelatinase MMP-9. Knock-down of MMP-9 and CTSS using siRNAs resulted in a synergistic suppression of MDA-MB-231 cell invasion, which was similarly observed with pharmacological inhibitors. During the screening of new drug candidates that suppress both CTSS and MMP-9, BJ-2302, a novel 7-azaindolin-2-one derivative, was discovered. Src, an upstream activator of both pathways (PI3K/Akt and Ras/Raf/ERK) responsible for the expression of CTSS and MMP-9, was identified as a high-affinity target of BJ-2302 (IC: 3.23 µM) through a Src kinase assay and a drug affinity responsive target stability (DARTS) assay. BJ-2302 effectively suppressed MDA-MB-231 cell invasion (Matrigel invasion assay) and metastasis (chorioallantoic membrane assay xenografted with MDA-MB-231-luc2-tdTomato cancer cells). Unlike Z-FL-COCHO (potent CTSS inhibitor), BJ-2302 did not induce any cytotoxicity in MCF-10A normal breast epithelial cells. Additionally, BJ-2302 (1 mg/kg) strongly suppressed TNBC cell proliferation in vitro and tumor growth in a xenograft mouse tumor model. The anti-metastatic and anti-tumor effects of BJ-2302 were superior to those of Z-FL-COCHO (1 mg/kg) or batimastat (30 mg/kg), a pan-MMP inhibitor. In summary, inhibition of Src kinase suppressed TNBC tumor growth and metastasis, and Src inhibitors such as BJ-2302 may constitute a novel therapeutic tool to treat breast cancer that expresses high levels of CTSS and MMP-9.
Topics: Aminopyridines; Animals; Cathepsins; Cell Proliferation; Cell Survival; Chickens; Chorioallantoic Membrane; Down-Regulation; Female; Humans; MCF-7 Cells; Matrix Metalloproteinase 9; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays; src-Family Kinases
PubMed: 30185799
DOI: 10.1038/s12276-018-0135-9 -
Oncotarget Apr 2018Primary human colorectal tumors with a high stromal content have an increased capacity to metastasize. Cancer-associated fibroblasts (CAFs) promote metastasis, but the...
Primary human colorectal tumors with a high stromal content have an increased capacity to metastasize. Cancer-associated fibroblasts (CAFs) promote metastasis, but the contribution of other stromal cell types is unclear. Here we searched for additional stromal cell types that contribute to aggressive tumor cell behavior. By making use of the 'immunome compendium'-a collection of gene signatures reflecting the presence of specific immune cell-types-we show that macrophage signatures are most strongly associated with a high CAF content and with poor prognosis in multiple large cohorts of primary tumors and liver metastases. Co-culturing macrophages with patient-derived colonospheres promoted 'budding' of small clusters of tumor cells from the bulk. Immunohistochemistry showed that budding tumor clusters in stroma-rich areas of T1 colorectal carcinomas were surrounded by macrophages. budding was accompanied by reduced levels of the tight junction protein occludin, but mRNA levels did not change, nor did markers of epithelial mesenchymal transition. Budding was accompanied by nuclear accumulation of β-catenin, which was also observed in budding tumor cell clusters . The NFκB inhibitor Sanguinarine resulted in a decrease in MMP7 protein expression and both NFκB inhibitor Sanguinarine and MMP inhibitor Batimastat prevented occludin degradation and budding. We conclude that macrophages contribute to the aggressive nature of stroma-rich colon tumors by promoting an MMP-dependent pathway that operates in parallel to classical EMT and leads to tight junction disruption.
PubMed: 29731961
DOI: 10.18632/oncotarget.24626 -
Toxins Jan 2017Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with...
BACKGROUND
Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, and , through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed.
METHODS
In this study, enzymatic toxins were functionally identified using a combined approach consisting of zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors.
RESULTS
Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with . nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14-18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays.
CONCLUSION
For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.
Topics: Animals; Chromatography, Liquid; Cnidarian Venoms; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Hemolysis; Hyaluronoglucosaminidase; Lipase; Metalloproteases; Molecular Weight; Protease Inhibitors; Proteomics; Scyphozoa; Sheep, Domestic; Tandem Mass Spectrometry
PubMed: 28134758
DOI: 10.3390/toxins9020047 -
Neuropsychopharmacology : Official... Oct 2016Estradiol (E2) perfusion rapidly increases the strength of fast excitatory transmission and facilitates long-term potentiation in the hippocampus, two effects likely...
Estradiol (E2) perfusion rapidly increases the strength of fast excitatory transmission and facilitates long-term potentiation in the hippocampus, two effects likely related to its memory-enhancing properties. Past studies showed that E2's facilitation of transmission involves activation of RhoA signaling leading to actin polymerization in dendritic spines. Here we report that brief exposure of adult male hippocampal slices to 1 nM E2 increases the percentage of postsynaptic densities associated with high levels of immunoreactivity for activated forms of the BDNF receptor TrkB and β1-integrins, two synaptic receptors that engage actin regulatory RhoA signaling. The effects of E2 on baseline synaptic responses were unaffected by pretreatment with the TrkB-Fc scavenger for extracellular BDNF or TrkB antagonism, but were eliminated by neutralizing antisera for β1-integrins. E2 effects on synaptic responses were also absent in conditional β1-integrin knockouts, and with inhibition of matrix metalloproteinases, extracellular enzymes that generate integrin ligands. We propose that E2, acting through estrogen receptor-β, transactivates synaptic TrkB and β1-integrin, and via mechanisms dependent on integrin activation and signaling, reversibly reorganizes the spine cytoskeleton and thereby enhances synaptic responses in adult hippocampus.
Topics: Animals; Animals, Newborn; Benzodioxoles; Dipeptides; Disks Large Homolog 4 Protein; Estrogen Receptor Modulators; Estrogens; Excitatory Postsynaptic Potentials; Female; Gene Expression Regulation; Guanylate Kinases; Hippocampus; Integrin beta1; Integrins; Male; Matrix Metalloproteinase Inhibitors; Membrane Proteins; Mice, Knockout; Neurons; Phenylalanine; Piperidines; Pyrazoles; Pyrimidines; Quinolines; Rats; Rats, Sprague-Dawley; Receptors, Estrogen; Thiophenes
PubMed: 27272766
DOI: 10.1038/npp.2016.83 -
Frontiers in Endocrinology 2019In the present study, we determined the cellular regulators of ERK1/2 and Akt signaling pathways in response to human CRF receptor (CRFR) activation in transfected COS-7...
In the present study, we determined the cellular regulators of ERK1/2 and Akt signaling pathways in response to human CRF receptor (CRFR) activation in transfected COS-7 cells. We found that Pertussis Toxin (PTX) treatment or sequestering Gβγ reduced CRFR-mediated activation of ERK1/2, suggesting the involvement of a G-linked cascade. Neither G/PKA nor G/PKC were associated with ERK1/2 activation. Besides, CRF induced EGF receptor (EGFR) phosphorylation at Tyr, and selective inhibition of EGFR kinase activity by AG1478 strongly inhibited the CRFR-mediated phosphorylation of ERK1/2, indicating the participation of EGFR transactivation. Furthermore, CRF-induced ERK1/2 phosphorylation was not altered by pretreatment with batimastat, GM6001, or an HB-EGF antibody indicating that metalloproteinase processing of HB-EGF ligands is not required for the CRF-mediated EGFR transactivation. We also observed that CRF induced Src and PYK2 phosphorylation in a Gβγ-dependent manner. Additionally, using the specific Src kinase inhibitor PP2 and the dominant-negative-SrcYF-KM, it was revealed that CRF-stimulated ERK1/2 phosphorylation depends on Src activation. PP2 also blocked the effect of CRF on Src and EGFR (Tyr) phosphorylation, further demonstrating the centrality of Src. We identified the formation of a protein complex consisting of CRFR, Src, and EGFR facilitates EGFR transactivation and CRFR-mediated signaling. CRF stimulated Akt phosphorylation, which was dependent on G/βγ subunits, and Src activation, however, was only slightly dependent on EGFR transactivation. Moreover, PI3K inhibitors were able to inhibit not only the CRF-induced phosphorylation of Akt, as expected, but also ERK1/2 activation by CRF suggesting a PI3K dependency in the CRFR ERK signaling. Finally, CRF-stimulated ERK1/2 activation was similar in the wild-type CRFR and the phosphorylation-deficient CRFR-Δ386 mutant, which has impaired agonist-dependent β-arrestin-2 recruitment; however, this situation may have resulted from the low β-arrestin expression in the COS-7 cells. When β-arrestin-2 was overexpressed in COS-7 cells, CRF-stimulated ERK1/2 phosphorylation was markedly upregulated. These findings indicate that on the base of a constitutive CRFR/EGFR interaction, the G/βγ subunits upstream activation of Src, PYK2, PI3K, and transactivation of the EGFR are required for CRFR signaling via the ERK1/2-MAP kinase pathway. In contrast, Akt activation via CRFR is mediated by the Src/PI3K pathway with little contribution of EGFR transactivation.
PubMed: 31920979
DOI: 10.3389/fendo.2019.00869 -
Breast Cancer Research and Treatment Nov 2017The CD44/CD24 cell phenotype is enriched in triple negative breast cancers, is associated with tumor invasive properties, and serves as a cell surface marker profile of...
PURPOSE
The CD44/CD24 cell phenotype is enriched in triple negative breast cancers, is associated with tumor invasive properties, and serves as a cell surface marker profile of breast cancer stem-like cells. Activation of Epidermal Growth Factor Receptor (EGFR) promotes the CD44/CD24 phenotype, but the specific signaling pathway downstream of EGFR responsible for this effect is not clear. The purpose of this study was to determine the role of the MEK/ERK pathway in the expansion of CD44/CD24 populations in TNBC cells in response to EGFR activation.
METHODS
Representative TNBC cell lines SUM159PT (claudin-low) and SUM149PT (basal) were used to evaluate cell surface expression of CD44 and CD24 by flow cytometry in response to EGFR and MEK inhibition or activation. EGFR and ERK phosphorylation levels were analyzed by Western blotting. The relationship between EGFR phosphorylation and MEK activation score in basal and claudin-low tumors from the TCGA database was examined.
RESULTS
Inhibition of ERK activation with selumetinib, a MEK1/2 inhibitor, blocked EGF-induced expansion of CD44/CD24 populations. Sustained activation of ERK by overexpression of constitutively active MEK1 was sufficient to expand CD44/CD24 populations in cells in which EGFR activity was blocked by either erlotinib, an EGFR kinase inhibitor, or BB-94, a metalloprotease inhibitor that prevents generation of soluble EGFR ligands. In basal and claudin-low tumors from the TCGA database, there was a positive correlation between EGFR_pY1068 and MEK activation score in tumors without genomic loss of DUSP4, a negative regulator of ERK, but not in tumors harboring DUSP4 deletion.
CONCLUSION
Our results demonstrate that ERK activation is a key event in EGFR-dependent regulation of CD44/CD24 populations. Furthermore, our findings highlight the role of ligand-mediated EGFR signaling in the control of MEK/ERK pathway output in TNBC tumors without DUSP4 loss.
Topics: Benzimidazoles; CD24 Antigen; Cell Line, Tumor; Databases, Genetic; Dual-Specificity Phosphatases; ErbB Receptors; Erlotinib Hydrochloride; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Mitogen-Activated Protein Kinase Phosphatases; Phenylalanine; Phosphorylation; Thiophenes; Triple Negative Breast Neoplasms
PubMed: 28791489
DOI: 10.1007/s10549-017-4440-0 -
The Journal of Parasitology Aug 2018In the course of a structure based drug discovery program the known anticancer candidate marimastat was uncovered as a potent inhibitor of an enzyme in nematode cuticle...
In the course of a structure based drug discovery program the known anticancer candidate marimastat was uncovered as a potent inhibitor of an enzyme in nematode cuticle biogenesis. It was shown to kill Caenorhabditis elegans, and the sheep parasites Haemonchus contortus and Teladorsagia circumcinta via an entirely novel nematode-specific pathway, specifically by inhibiting cuticle-remodelling enzymes that the parasites require for the developmentally essential moulting process. This discovery prompted an investigation of the compound's effect on Heligmosomoides polygyrus parasites in a mouse model of helminth infection. Mice were administered the drug via oral gavage daily from day of infection for a period of 2 wk. A second group received the drug via intra-peritoneal implantation of an osmotic minipump for 4 wk. Control groups were administered identical volumes of water by oral gavage in both cases. Counts of H. polygyrus faecal egg and larval load showed that marimastat effected a consistent and significant reduction in egg laying, and a consistent but minor reduction in adult worm load when administered every day, starting on the first day of infection. However, the drug failed to have any significant effect on egg counts or worm burdens when administered to mice with established infections. Therefore, marimastat does not appear to show promise as an anthelmintic in gastrointestinal nematode infections, although other metalloproteases such as batimastat may prove more effective.
PubMed: 30085900
DOI: 10.1645/18-33 -
The Journal of Biological Chemistry Mar 2015ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously...
ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1' position. Cys(392), present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys(392) for a Ser increased the reactivity with α2-macroglobulin but not with casein or Cm-Tf. Substitution of Asp(362) for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors.
Topics: ADAM Proteins; Amino Acid Sequence; Catalytic Domain; Crystallography, X-Ray; Gene Expression Regulation, Enzymologic; Humans; Metalloproteases; Protein Structure, Tertiary; Proteolysis; Substrate Specificity; Tissue Inhibitor of Metalloproteinases; Zinc
PubMed: 25564618
DOI: 10.1074/jbc.M114.601724 -
Nature Communications Jun 2019Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in...
Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients.
Topics: Adipocytes; Adipogenesis; Adipose Tissue; Adolescent; Age of Onset; Animals; Annexin A2; Case-Control Studies; Dysferlin; Elapid Venoms; Female; Fibrosis; Humans; In Vitro Techniques; Male; Mice; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Phenylalanine; Protease Inhibitors; Severity of Illness Index; Stem Cells; Thiophenes; Young Adult
PubMed: 31160583
DOI: 10.1038/s41467-019-10438-z -
Frontiers in Cardiovascular Medicine 2023Skeletal muscle injury in peripheral artery disease (PAD) has been attributed to vascular insufficiency, however evidence has demonstrated that muscle cell responses...
Skeletal muscle injury in peripheral artery disease (PAD) has been attributed to vascular insufficiency, however evidence has demonstrated that muscle cell responses play a role in determining outcomes in limb ischemia. Here, we demonstrate that genetic ablation of Pax7 muscle progenitor cells (MPCs) in a model of hindlimb ischemia (HLI) inhibited muscle regeneration following ischemic injury, despite a lack of morphological or physiological changes in resting muscle. Compared to control mice (Pax7), the ischemic limb of Pax7-deficient mice (Pax7) was unable to generate significant force 7 or 28 days after HLI. A significant increase in adipose was observed in the ischemic limb 28 days after HLI in Pax7 mice, which replaced functional muscle. Adipogenesis in Pax7 mice corresponded with a significant increase in PDGFRα fibro/adipogenic progenitors (FAPs). Inhibition of FAPs with batimastat decreased muscle adipose but increased fibrosis. , Pax7 MPCs failed to form myotubes but displayed increased adipogenesis. Skeletal muscle from patients with critical limb threatening ischemia displayed increased adipose in more ischemic regions of muscle, which corresponded with fewer satellite cells. Collectively, these data demonstrate that Pax7 MPCs are required for muscle regeneration after ischemia and suggest that muscle regeneration may be an important therapeutic target in PAD.
PubMed: 36937923
DOI: 10.3389/fcvm.2023.1118738