Smad7 has been identified as a negative regulator of transforming growth factor  (TGF-) signaling by interfering with the phosphorylation of other Smad proteins by TGF- receptor type I (TRI). We established a mink lung epithelial (Mv1Lu) cell line where ectopic expression of Smad7 is tightly controlled by doxycycline using an improved Tet-on system. Once induced by doxycycline, the recombinant Smad7 was localized predominantly in the perinuclear region and in the cytoplasm. However, the type of culture surface alters the subcellular localization of Smad7: on plastic or on fibronectin-coated glass, Smad7 was localized in the cytoplasm; but when the cells were cultured on glass, nuclear localization was observed. TGF- stimulation did not alter substantially the cellular distribution of Smad7. Importantly, the expression of recombinant Smad7 differentially inhibited TGF- signaling pathways. Consistent with previous studies, Smad7 inhibited TGF--stimulated induction of type 1 plasminogen activator inhibitor as measured by p3TP-Lux reporter. However, expression of Smad7 had little effect on TGF--induced growth inhibition.
Transforming Growth Factor-β (TGF-β) and Epidermal Growth Factor (EGF) signaling pathways are both independently implicated as key regulators in tumor formation and progression. Here, we report that the tumor-associated overexpression of epidermal growth factor receptor (EGFR) desensitizes TGF-β signaling and its cytostatic regulation through specific and persistent Stat3 activation and Smad7 induction in vivo. In human tumor cell lines, reduction of TGF-β-mediated Smad2 phosphorylation, nuclear translocation and Smad3 target gene activation were observed when EGFR was overexpressed, but not in cells that expressed EGFR at normal levels. We identified Stat3, which is activated specifically and persistently by overexpressed EGFR, as a key signaling molecule responsible for the reduced TGF-β sensitivity. Stable knockdown of Stat3 using small hairpin RNA(shRNA) in Head and Neck (HN5) and Epidermoid (A431) tumor cell lines resulted in reduced growth compared with control shRNA-transfected cells when grown as subcutaneous tumor xenografts. Furthermore, xenografts with Stat3 knockdown displayed increased Smad3 transcriptional activity, increased Smad2 phosphorylation and decreased Smad7 expression compared with control xenografts in vivo. Consistently, Smad7 mRNA and protein expression was also significantly reduced when EGFR activity was blocked by a specific tyrosine kinase inhibitor, AG1478, or in Stat3 knockdown tumors. Similarly, Smad7 knockdown also resulted in enhanced Smad3 transcriptional activity in vivo. Importantly, there was no uptake of subcutaneous HN5 xenografts with Smad7 knockdown. Taken together, we demonstrate here that targeting Stat3 or Smad7 for knockdown results in resensitization of TGF-β's cytostatic regulation in vivo. Overall, these results establish EGFR/Stat3/Smad7/TGF-β signaling axis driving tumor growth, which can be targeted therapeutically.
Background Bone morphogenetic proteins (BMPs) have been reported to maintain epithelial integrity and to antagonize the transforming growth factor β (TGFβ)-induced epithelial to mesenchymal transition. The expression of soluble BMP antagonists is dysregulated in cancers and interrupts proper BMP signaling in breast cancer. Methods In this study, we mined the prognostic role of BMP antagonists GREMLIN 1 (GREM1) in primary breast cancer tissues using in-house and publicly available datasets. We determined which cells express GREM1 RNA using in situ hybridization (ISH) on a breast cancer tissue microarray. The effects of Grem1 on the properties of breast cancer cells were assessed by measuring the mesenchymal/stem cell marker expression and functional cell-based assays for stemness and invasion. The role of Grem1 in breast cancer-associated fibroblast (CAF) activation was measured by analyzing the expression of fibroblast markers, phalloidin staining, and collagen contraction assays. The role of Grem1 in CAF-induced breast cancer cell intravasation and extravasation was studied by utilizing xenograft zebrafish breast cancer (co-) injection models. Results Expression analysis of clinical breast cancer datasets revealed that high expression of GREM1 in breast cancer stroma is correlated with a poor prognosis regardless of the molecular subtype. The large majority of human breast cancer cell lines did not express GREM1 in vitro, but breast CAFs did express GREM1 both in vitro and in vivo. Transforming growth factor β (TGFβ) secreted by breast cancer cells, and also inflammatory cytokines, stimulated GREM1 expression in CAFs. Grem1 abrogated bone morphogenetic protein (BMP)/SMAD signaling in breast cancer cells and promoted their mesenchymal phenotype, stemness, and invasion. Moreover, Grem1 production by CAFs strongly promoted the fibrogenic activation of CAFs and promoted breast cancer cell intravasation and extravasation in co-injection xenograft zebrafish models. Conclusions Our results demonstrated that Grem1 is a pivotal factor in the reciprocal interplay between breast cancer cells and CAFs, which promotes cancer cell invasion. Targeting Grem1 could be beneficial in the treatment of breast cancer patients with high Grem1 expression.
Granulocyte colony-stimulating factor (G-CSF) initiates its effects on cells of the neutrophil lineage by inducing formation of a homodimeric receptor complex. The structure of the G-CSF receptor has not yet been determined, therefore we used molecular modeling to identify regions of the receptor that were likely to be involved in ligand binding. The G-CSF receptor sequence was aligned with all the available sequences of the gp130 and growth hormone receptor families and a model of the cytokine receptor homologous domain was constructed, based on the growth hormone receptor structure. Alanine substitution mutagenesis was performed on loops and individual residues that were predicted to bind ligand. Mutant receptors were expressed in factor-dependent Ba/F3 cells and assessed for proliferation response and ligand binding. Six residues were identified that significantly reduced receptor function, with Arg 288 in the F-G loop having the greatest effect. These residues formed a binding face on the receptor model resembling the growth hormone receptor site, which suggests that the model is reasonable. However, electrostatic analysis of the model provided further evidence that the mechanism of receptor dimerization is different from that of the growth hormone receptor. The granulocyte colony-stimulating factor receptor (G-CSF-R)1 is a member of the class 1 cytokine receptor family which is defined by the presence of a cytokine receptor homology (CRH) domain that is characterized by four conserved Cys residues and a conserved WSXWS (Trp-Ser-X-Trp-Ser, where X is any amino acid) sequence (1). The receptor is expressed predominantly on cells of the neutrophil lineage (2), hence interaction with G-CSF stimulates proliferation and differentiation of neutrophils from precursor cells (3). G-CSF is required for normal neutrophil production in vivo (4). The molecular mechanisms of receptor activation and signal transduction are at present poorly understood. The receptor is most probably homodimerized by ligand binding (5-7), resulting in activation of cytoplasmic signaling pathways including the Jak-STAT pathway and the MAP kinase pathway (reviewed by Avalos (8)).Bazan (1) proposed that the conserved CRH domain is comprised of two modules of fibronectin type III (FnIII)-like structure, each containing seven -strands which form two -sheets. This model has been confirmed by determination of the crystal structures of several receptors including the growth hormone receptor (GH-R) (9), prolactin receptor (10), erythropoietin receptor (EPO-R) (11), and related class 2 cytokine receptors: tissue factor (12) and interferon-␥ receptor ␣ (13). The highly conserved Cys residues and WSXWS sequence are likely to be required for maintenance of the structure of the CRH domain. In the G-CSF-R, each of the CRH domain modules (termed BN and BC) have been expressed as soluble proteins, both of which bind G-CSF with low affinity (14, 15). In both the BN and BC domains, the disulfide bonds were critical for maintaining a stably folded protein and...
To define regions of the granulocyte colony-stimulating factor (G-CSF) receptor that are important for ligand binding, neutralising monoclonal antibodies to the human receptor have been produced. Eleven antibodies recognised six different receptor epitopes. Antibodies from three of the epitope groups were able to detect the receptor by western blotting but did not inhibit G-CSF binding. The other three antibody groups inhibited G-CSF binding either completely (groups 1 and 2) or partially (group 3). All the antibodies inhibited proliferation of BA/F3 cells expressing the G-CSF receptor to varying extents. By using human-marine chimeric receptors, the binding sites of the antibodies were mapped to the immunoglobulin-like domain (groups 1 and 3), the cytokine receptor homologous domain (group 2) or the fibronectin type III domains (groups 4 to 6). These results show that the immunoglobulin-like and cytokine receptor homologous domains of the receptor are important for ligand binding and subsequent signalling.
The epidermal growth factor receptor (EGFR) plays an important role in many types of human cancers. Receptor amplification, autocrine activation and/or deletion of exons 2-7 of EGFR gene have all been associated with tumor development. The traditional model of EGFR activation via ligand induced dimerization and consequential kinase activation does not provide full understanding of its tumorigenicity. The main function of the receptor extracellular domain (ECD) has been thought to be ligand recognition and binding. We report that the EGFR ECD, through its association also negatively regulates the activity of the intracellular kinase in the absence of ligand. Even in the absence of its ligands, the EGF receptor forms homodimers, however, the ECD prevents constitutive receptor kinase activation through its intrinsic ligand-independent interaction. The removal of this domain, either partial or total, results in constitutive activation of the receptor kinase as observed by its phosphorylation in intact cells. Furthermore, EGF receptors truncated in the ECD induce phosphorylation of the wild-type full-length receptor, indicating an inter-molecular inhibitory mechanism by the receptor ECD. The tumor associated delta2-7EGFR mutant also dimerizes with and phosphorylates the wild type EGFR in the absence of ligand. Thus, in addition to its role in ligand recognition, EGFR ECD interacts with each other, imposing an inhibitory effect on the activation of the intracellular kinase.
BackgroundMetastasis is a process where only a small subset of cells is capable of successfully migrating to and propagating at secondary sites. TGF-β signalling is widely known for its role in cancer metastasis and is associated with cell migration in whole cell populations.FindingsWe extend these findings by investigating the role of TGF-β signalling in promoting migration and motility by imaging the signalling activity in live, individual MDA-MB-231 cancer cells utilizing a novel Smad3 Td-Tomato reporter adenovirus. Here we find that not all MDA-MB-231 cancer cells have similar TGF-β mediated Smad3 transcription activity and display at least two distinct migratory populations. Importantly, Smad3 activity was significantly higher within migratory cells compared to non-migrated cells in wound healing and transwell assays. Furthermore, time-lapse experiments showed that MDA-MB-231 cells displaying Smad3 activity moved faster and a greater distance compared to cells not displaying Smad3 reporter activity. Interestingly, despite being more motile than cells with undetectable levels of Smad3 activity, high Smad3 activity was detrimental to cell motility compared to low and medium level of Smad3 activity.ConclusionsWe have developed a method enabling real-time visualization of TGF-β signalling in single live cells. Breast cancer cell motility and migration is driven by sub-populations of cells with dynamic TGF-β-Smad3 activity. Those sub-populations may be responsible for tumor invasion and metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0309-1) contains supplementary material, which is available to authorized users.
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