TGF-β Co-receptor Research Articles

Expression of endoglin, a TGFβ co-receptor, not ALK5 correlates with the ability of passaged human articular chondrocytes to form cartilage tissue in vitro

Expression of endoglin, a TGFβ co-receptor, not ALK5 correlates with the ability of passaged human articular chondrocytes to form cartilage tissue in vitro

Increased type III TGF-β receptor shedding decreases tumorigenesis through induction of epithelial-to-mesenchymal transition.

The type III TGF-β receptor (TβRIII) is a TGF-β co-receptor that presents ligand to the type II TGF-β receptor to initiate signaling. TβRIII also undergoes ectodomain shedding to release a soluble form (sTβRIII) that can bind ligand, sequestering it away from cell surface receptors. We have previously identified a TβRIII extracellular mutant that has enhanced ectodomain shedding (“super shedding (SS)” – TβRIII-SS). Here we utilize TβRIII-SS to study the balance of cell surface and soluble TβRIII in the context of lung cancer. We demonstrate that expressing TβRIII-SS in lung cancer cell models induces epithelial-to-mesenchymal transition (EMT) and that these TβRIII-SS (EMT) cells are less migratory, invasive and adhesive and more resistance to gemcitabine. Moreover, TβRIII-SS (EMT) cells exhibit decreased tumorigenicity but increased growth rate in vitro and in vivo. These studies suggest that the balance of cell surface and soluble TβRIII may regulate a dichotomous role for TβRIII during cancer progression.

NOX4/NADPH oxidase regulates the expression of endoglin, a TGF-β co-receptor

NOX4/NADPH oxidase regulates the expression of endoglin, a TGF-β co-receptor

Highlights of This Issue

Raja and colleagues explored changes in levels of ctDNA in advanced/metastatic NSCLC and UC patients receiving the anti-PD-L1 monoclonal antibody durvalumab. Reduction in ctDNA levels at 6 weeks was associated with RECIST responses, PFS and OS, and often preceded RECIST response. Thus, early changes in ctDNA may predict long-term benefit from immunotherapy and enable treatment decisions, particularly in indications in which assessing radiographic responses may be difficult.Mesothelioma is an aggressive cancer with limited treatment options and often with resistance to immunotherapy. To overcome resistance to previous treatments, Tallón de Lara and colleagues showed that the synergy of gemcitabine and immune checkpoint inhibitors (ICI) outperformed single treatment in a preclinical mouse model of mesothelioma. Two patients with mesothelioma, who did not respond to the single treatments, responded to the combination when given. Interestingly, dexamethasone, widely used in the clinics to treat side effects, dampened the response in the preclinical model. This study gives a new rationale to combine ICI and gemcitabine in mesothelioma patientsPatients with leukemia can develop bone defects (osteonecrosis) in hip bones, which can lead to joint collapse. A new treatment involves drilling a track in the affected bone (decompression surgery), aspirating marrow cells from the pelvis and implanting them into the bone defect. To visualize the cells, the authors treated patients with an iron supplement 1 to 2 days before the surgery. The cells in the bone marrow took up the iron. When iron-loaded cells were then harvested from the bone marrow and transplanted in the bone defect, they could be detect with MR imaging. This new stem cell imaging approach may help to better understand and optimize stem cell therapies.TRC105 is a neutralizing antibody targeting the TGF-β coreceptor endoglin and is currently being tested in clinical trials as antiangiogenic therapy. TRC105 prevents binding of BMP-9 to endoglin, which controls (tumor) angiogenesis. Paauwe and colleagues show that endoglin also is expressed on cancer-associated fibroblasts (CAFs) and reveal a role for this receptor in CAF invasion. TRC105 treatment reduced invasive capacity of CAFs in vitro and metastatic spread of CRC in vivo. This underlines the potential of TRC105 to be more than a “classic anti-angiogenic drug,” targeting both endothelium and CAF, thereby limiting metastatic spread on multiple levels.

The Depletion of TGF-β Co-Receptor CD109 Induces Erythroid Differentiation of TF-1 Cells: A Model of Preferential Commitment of PIGA-Mutated Hematopoietic Stem Cells in Immune-Mediated Bone Marrow Failure

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may play an important role in the regulation of the HSPC commitment, given the fact that a lack of GPI-APs due to PIGA mutations allows HSPCs to preferentially undergo commitment into mature blood cells under immune pressure against HSPCs in patients with acquired aplastic anemia. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs has not been clarified. TF-1 is one of a few myeloid leukemia cell lines that express CD109, the proliferation of which is dependent on GM-CSF. Since TF-1 undergoes erythroid differentiation in response to δ-5-aminolevulinic acid (δ-ALA), and its differentiation is reportedly inhibited by TGF-β, a lack of GPI-APs due to PIGA mutation and/or the knockout (KO) of CD109 may affect the differentiation of TF-1 cells. [Objectives/Methods] To gain insights into the role of GPI-APs on HSPCs, we established a PIGA-mutated TF-1 cell line by culturing TF-1 in the presence of α-toxin for several months, and a CD109 KO TF-1 cell line using a CRISPR-Cas 9 system. The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) on TF-1 cells using flow cytometry (FCM) and iron staining. We also determined the CD109 expression by HSPCs from healthy individuals and C57BL/6 mice using FCM and a quantitative PCR. [Results] Both GPI-AP-deficient TF-1 cells that had a PIGA mutation (7 nucleotide deletion at position 291-297 [TTGTCAC] in exon 2) and CD109 KO TF-1 cells showed slower proliferation than wild-type (WT) TF-1 cells. Similarly to TF-1 cells treated with δ-ALA, both mutant cells expressed GPA, exhibited erythroid morphology, and were positive for iron granules, suggesting that GPI-APs inhibited the erythroid differentiation of WT TF-1 cells that were cultured in RPMI1640 containing 10% fetal bovine serum (FBS), and that the GPI-AP that plays a key role in the inhibition of erythroid differentiation is CD109. Since low levels (1-2 ng/ml) of TGF-β in the serum-containing culture medium were suspected to inhibit the erythroid differentiation of WT TF-1 through its binding to CD109, WT TF-1 cells were cultured in a serum-free medium Expi293 Expression Medium for 10 days. While control TF-1 cells cultured in the serum-containing RPMI1640 were negative for the expression of GPA, 77.0-84.5% of the cultured TF-1 cells expressed GPA and exhibited erythroid morphology. CD109 was expressed by 12.1-18.3% of CD34+CD38- cells, 4.5-7.4% of common myeloid progenitor cells (CMPs), 20.8-42.4% of megakaryocyte-erythrocyte progenitor cells (MEPs), and 14.2-22.0% of granulocyte macrophage progenitor cells (GMPs) in the bone marrow of healthy individuals, while murine CD48-CD150+CD34- LSK cells were negative for either CD109 protein or mRNA. [Conclusions] CD109 protects TF-1 cells from differentiating into erythroid cells in serum-containing culture. In contrast to keratinocytes, the CD109 on TF-1 cells, and possibly on HSPCs, may enhance TGF-β signaling, and the lack of the GPI-AP might make PIGA-mutated HSPCs insensitive to TGF-β, leading to the preferential commitment of mutant HSPCs to mature blood cells in immune-mediated bone marrow failure. [Display omitted] DisclosuresNakao:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Characterization and Drug Sensitivity of a New High-Grade Myxofibrosarcoma Cell Line.

Myxofibrosarcoma (MFS) belongs to the group of sarcoma tumors, which represent only 1% of the totality of adult tumors worldwide. Thus, given the rare nature of this cancer, this makes the availability of MFS cell lines difficult. In an attempt to partially fill this gap, we immortalized a primary culture of MFS (IM-MFS-1) and compared the cell morphology with patient’s tumor tissue. IM-MFS-1 was genetically characterized through a Comparative Genomic Hybridization (CGH) array and the mesenchymal phenotype was evaluated using Polymerase chain reaction (PCR) and immunofluorescence staining. Drug sensitivity for MFS therapies was monitored over time in cultures. We confirmed the conservation of the patient’s tumor cell morphology and of the mesenchymal phenotype. Conversely, the synthesis and expression of CD109, a TGFβ co-receptor used to facilitate the diagnosis of high-grade MFS diagnosis, was maintained constant until high cancer cell line passages. The CGH array revealed a complex karyotype with cytogenetic alterations that include chromosome regions associated with genes involved in tumor processes. Cytotoxicity assays show drug sensitivity constantly increased during the culture passages until a plateau was reached. In conclusion, we established and characterized a new MFS cell line that can be used for future preclinical and molecular studies on soft tissue sarcomas.

Bone Morphogenetic Protein 9 Reduces Cardiac Fibrosis and Improves Cardiac Function in Heart Failure.

Heart failure is a growing cause of morbidity and mortality worldwide. Transforming growth factor beta (TGF-β1) promotes cardiac fibrosis, but also activates counterregulatory pathways that serve to regulate TGF-β1 activity in heart failure. Bone morphogenetic protein 9 (BMP9) is a member of the TGFβ family of cytokines and signals via the downstream effector protein Smad1. Endoglin is a TGFβ coreceptor that promotes TGF-β1 signaling via Smad3 and binds BMP9 with high affinity. We hypothesized that BMP9 limits cardiac fibrosis by activating Smad1 and attenuating Smad3, and, furthermore, that neutralizing endoglin activity promotes BMP9 activity. We examined BMP9 expression and signaling in human cardiac fibroblasts and human subjects with heart failure. We used the transverse aortic constriction-induced model of heart failure to evaluate the functional effect of BMP9 signaling on cardiac remodeling. BMP9 expression is increased in the circulation and left ventricle (LV) of human subjects with heart failure and is expressed by cardiac fibroblasts. Next, we observed that BMP9 attenuates type I collagen synthesis in human cardiac fibroblasts using recombinant human BMP9 and a small interfering RNA approach. In BMP9-/- mice subjected to transverse aortic constriction, loss of BMP9 activity promotes cardiac fibrosis, impairs LV function, and increases LV levels of phosphorylated Smad3 (pSmad3), not pSmad1. In contrast, treatment of wild-type mice subjected to transverse aortic constriction with recombinant BMP9 limits progression of cardiac fibrosis, improves LV function, enhances myocardial capillary density, and increases LV levels of pSmad1, not pSmad3 in comparison with vehicle-treated controls. Because endoglin binds BMP9 with high affinity, we explored the effect of reduced endoglin activity on BMP9 activity. Neutralizing endoglin activity in human cardiac fibroblasts or in wild-type mice subjected to transverse aortic constriction-induced heart failure limits collagen production, increases BMP9 protein levels, and increases levels of pSmad1, not pSmad3. Our results identify a novel functional role for BMP9 as an endogenous inhibitor of cardiac fibrosis attributable to LV pressure overload and further show that treatment with either recombinant BMP9 or disruption of endoglin activity promotes BMP9 activity and limits cardiac fibrosis in heart failure, thereby providing potentially novel therapeutic approaches for patients with heart failure.

PO-459 Unravelling endoglin as a potential therapeutic target for the treatment of uveal melanoma

IntroductionUveal Melanoma(UM) is a rare deadly tumour where, despite highly aggressive management of the primary disease, approximately 50% of patients develop metastases. Therapies directed towards systemic/metastatic disease have failed in the clinic.Endoglin (ENG),a co-receptor of TGF-β,plays an important role in tissue remodelling and cancer.A soluble form is produced upon proteolytic cleavage by matrix metalloprotease MMP14.Our work focuses on the role of ENG/MMP14, how they influence UM metastasis, and their potential as therapeutic targets.Material and methodsNormalised gene expression data with patient follow-up from 80 primary UM tumours was downloaded from The Cancer Genome Atlas (TCGA) NIH Data Portal.mRNA/protein from UM and Ewing Sarcoma cell lines were isolated, and ENG/MMP14 levels were evaluated by q-RT-PCR/western blot. Immunohistochemistry analysis of ENG/MMP14 expression was performed in 2 independent patient cohorts.MTT assays were performed to evaluate proliferation inhibition after exposure to OMTX503 anti-ENG Antibody Drug Conjugate (ADC).CRISPR technology with high fidelity Cas9 was used to knockout MMP14/ENG, which was validated by mismatch-cleavage assays and by the loss of protein expression.Results and discussionsBioinformatics analysis showed that a higher expression of ENG correlated with worst disease-free survival (DFS)(p=0.00023), whereas MMP14 showed a similar trend without reaching statistical significance(p=0.077).These results differ from skin melanoma DFS(p=0.74 for ENG,p=0.22 for MMP14).Pathway enrichment analysis showed that higher ENG expression is associated with a higher abundance of CD8+T-cells (p<0.002),endothelial cells(p<0.003) and fibroblasts(p<0.001).In the 2 patient cohorts, differential levels of ENG/MMP14 were detected.In vitro, cell line MUM2B expressed higher levels of ENG/MMP14.UM cell lines were highly sensitive to treatment with ADC OMTX503, with IC50 in the sub-nM range.Sensitivity was dependent on ENG expression.Knockout of MMP14 resulted in lack of protein expression in the single cell derived clones.The effect on ENG shedding and malignant features upon lack of MMP14 is currently being analysed.ConclusionENG/MMP14 are expressed in UM cell lines and in patient tumour samples.Results suggest that higher levels of ENG are associated with a more aggressive phenotype.ENG is an attractive target, and ENG-targeting biologics such as OMTX503 show promising in vitro activity.In-depth evaluation of the role of ENG/MMP14 in UM is ongoing.

Endothelial cell biology of Endoglin in hereditary hemorrhagic telangiectasia.

Mutations in the Endoglin (Eng) gene, an auxiliary receptor in the transforming growth factor beta (TGFβ)-superfamily signaling pathway, are responsible for the human vascular disorder hereditary hemorrhagic telangiectasia (HHT) type 1, characterized in part by blood vessel enlargement. A growing body of work has uncovered an autonomous role for Eng in endothelial cells. We will highlight the influence of Eng on distinct cellular behaviors, such as migration and shape control, which are ultimately important for the assignment of proper blood vessel diameters. How endothelial cells establish hierarchically ordered blood vessel trees is one of the outstanding questions in vascular biology. Mutations in components of the TGFβ-superfamily of signaling molecules disrupt this patterning and cause arteriovenous malformations (AVMs). Eng is a TGFβ coreceptor enhancing signaling through the type I receptor Alk1. Recent studies identified bone morphogenetic proteins (BMPs) 9 and 10 as the primary ligands for Alk1/Eng. Importantly, Eng potentiated Alk1 pathway activation downstream of hemodynamic forces. New results furthermore revealed how Eng affects endothelial cell migration and cell shape control in response to these forces, thereby providing new avenues for our understanding of AVM cause. We will discuss the interplay of Eng and hemodynamic forces, such as shear stress, in relation to Alk1 receptor activation. We will furthermore detail how this signaling pathway influences endothelial cell behaviors important for the establishment of hierarchically ordered blood vessel trees. Finally, we will provide an outlook how these insights might help in developing new therapies for the treatment of HHT.

Role of TGF-β co-receptor CD109 in extracellular matrix production of smooth muscle cells

CD109 is a glycosylphosphatidylinositol (GPI)-linked glycoprotein located on the plasma membrane and has no intracellular domain. It has been reported that it attenuates the TGF-β signaling in skin fibroblastes and cancer cells by competitive binding to TGF-β factors and promoting internalization and degradation of TGF-β receptors. We found less production of extracellular matrix (ECM) and higher expression of CD109 in the carotids of angiotensin II-treated mice in which integrin alphaV is inactivated specifically in smooth muscle cells (SMC). We want to study the role of CD109 on the production of extracellular matrix proteins in primary SMC. CD109 is overexpressed by expressing vector (in collaboration with Dr. Murakumo Y.) or decreased by CD109 siRNA in SMC that is analyzed by the cellular and molecular methods. Our results showed that the overexpression of full-length form or soluble form of CD109 attenuated the production of ECM proteins such as collagens and fibronectin in SMC, in contrast with the situation in which the CD109 is knock-downed by siRNA. The response to TGF-β1 and angiotensin II treatment is also attenuated in CD109 overexpression SMC and enhanced in CD109 knock-down SMC. The signaling pathways (phosphorylation of ERK, Akt, Smad2/3, STAT3) related to CD109 are under the investigation. The promoter of CD109 is also under the study in order to understand the mechanism leading to upregulation of CD109 in response to angiotensin II treatment in the integrin aphaV SMC-KO mice. CD109 receptor could be one important regulator of vascular fibrosis and potential target for anti-fibrosis treatment.

Endoglin haploinsufficiency is associated with differential regulation of extracellular matrix production during skin fibrosis and cartilage repair in mice.

Transforming growth factor (TGF)-β is a multifunctional growth factor with potent pro-fibrotic effects. Endoglin is a TGF-β co-receptor that strongly regulates TGF-β signaling in a variety of cell types. Although aberrant regulation of TGF-β signaling is known to play a key role in fibrotic diseases such as scleroderma and impaired cartilage repair, the significance of endoglin function in regulating these processes is poorly understood. Here we examined whether endoglin haploinsufficiency regulates extracellular (ECM) protein expression and fibrotic responses during bleomycin induced skin fibrosis and surgically induced osteoarthritis, using endoglin-heterozygous (Eng+/-) mice and wild-type (Eng+/+) littermates. Skin fibrosis was induced by injecting mice intradermally with bleomycin or vehicle. Osteoarthritis was induced surgically by destabilization of medial meniscus. Dermal thickness, cartilage integrity and ECM protein expression were then determined. Eng+/- mice subjected to bleomycin challenge show a marked decrease in dermal thickness (P < 0.005) and reduced collagen content and decreased collagen I, fibronectin, alpha-smooth muscle actin levels as compared to Eng+/+ mice, both under basal and bleomycin treated conditions. Eng+/- mice undergoing surgically induced osteoarthritis show no differences in the degree of cartilage degradation, as compared to Eng+/+ mice, although chondrocytes isolated from Eng+/- display markedly enhanced collagen II levels. Our findings suggest that endoglin haploinsufficiency in mice ameliorates bleomycin-induced skin fibrosis suggesting that endoglin represents a pro-fibrotic factor in the mouse skin. However, endoglin haploinsufficiency does not protect these mice from surgically indicedcartilage degradation, demonstrating differential regulation of endoglin action during skin and cartilage repair.

Endoglin interacts with VEGFR2 to promote angiogenesis.

Endoglin, a TGF-β coreceptor predominantly expressed in endothelial cells, plays an important role in vascular development and tumor-associated angiogenesis. However, the mechanism by which endoglin regulates angiogenesis, especially during tip cell formation, remains largely unknown. In this study, we report that endoglin promoted VEGF-induced tip cell formation. Mechanistically, endoglin interacted with VEGF receptor (VEGFR)-2 in a VEGF-dependent manner, which sustained VEGFR2 on the cell surface and prevented its degradation. Endoglin mutants deficient in the ability to interact with VEGFR2 failed to sustain VEGFR2 on the cell surface and to promote VEGF-induced tip cell formation. Further, an endoglin-targeting monoclonal antibody (mAb), TRC105, cooperated with a VEGF-A targeting mAb, bevacizumab, to inhibit VEGF signaling and tip cell formation in vitro and to inhibit tumor growth, metastasis, and tumor-associated angiogenesis in a murine tumor model. This study demonstrate a novel mechanism by which endoglin initiates and regulates VEGF-driven angiogenesis while providing a rationale for combining anti-VEGF and anti-endoglin therapy in patients with cancer.-Tian, H., Huang, J. J., Golzio, C., Gao, X., Hector-Greene, M., Katsanis, N., Blobe, G. C. Endoglin interacts with VEGFR2 to promote angiogenesis.

Inhibiting DPP4 in a mouse model of HHT1 results in a shift towards regenerative macrophages and reduces fibrosis after myocardial infarction.

AimsHereditary Hemorrhagic Telangiectasia type-1 (HHT1) is a genetic vascular disorder caused by haploinsufficiency of the TGFβ co-receptor endoglin. Dysfunctional homing of HHT1 mononuclear cells (MNCs) towards the infarcted myocardium hampers cardiac recovery. HHT1-MNCs have elevated expression of dipeptidyl peptidase-4 (DPP4/CD26), which inhibits recruitment of CXCR4-expressing MNCs by inactivation of stromal cell-derived factor 1 (SDF1). We hypothesize that inhibiting DPP4 will restore homing of HHT1-MNCs to the infarcted heart and improve cardiac recovery.Methods and resultsAfter inducing myocardial infarction (MI), wild type (WT) and endoglin heterozygous (Eng+/-) mice were treated for 5 days with the DPP4 inhibitor Diprotin A (DipA). DipA increased the number of CXCR4+ MNCs residing in the infarcted Eng+/- hearts (Eng+/- 73.17±12.67 vs. Eng+/- treated 157.00±11.61, P = 0.0003) and significantly reduced infarct size (Eng+/- 46.60±9.33% vs. Eng+/- treated 27.02±3.04%, P = 0.03). Echocardiography demonstrated that DipA treatment slightly deteriorated heart function in Eng+/- mice. An increased number of capillaries (Eng+/- 61.63±1.43 vs. Eng+/- treated 74.30±1.74, P = 0.001) were detected in the infarct border zone whereas the number of arteries was reduced (Eng+/- 11.88±0.63 vs. Eng+/- treated 6.38±0.97, P = 0.003). Interestingly, while less M2 regenerative macrophages were present in Eng+/- hearts prior to DipA treatment, (WT 29.88±1.52% vs. Eng+/- 12.34±1.64%, P<0.0001), DPP4 inhibition restored the number of M2 macrophages to wild type levels.ConclusionsIn this study, we demonstrate that systemic DPP4 inhibition restores the impaired MNC homing in Eng+/- animals post-MI, and enhances cardiac repair, which might be explained by restoring the balance between the inflammatory and regenerative macrophages present in the heart.

LRP1 integrates murine macrophage cholesterol homeostasis and inflammatory responses in atherosclerosis.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor with diverse physiological roles, ranging from cellular uptake of lipoproteins and other cargo by endocytosis to sensor of the extracellular environment and integrator of a wide range of signaling mechanisms. As a chylomicron remnant receptor, LRP1 controls systemic lipid metabolism in concert with the LDL receptor in the liver, whereas in smooth muscle cells (SMC) LRP1 functions as a co-receptor for TGFβ and PDGFRβ in reverse cholesterol transport and the maintenance of vascular wall integrity. Here we used a knockin mouse model to uncover a novel atheroprotective role for LRP1 in macrophages where tyrosine phosphorylation of an NPxY motif in its intracellular domain initiates a signaling cascade along an LRP1/SHC1/PI3K/AKT/PPARγ/LXR axis to regulate and integrate cellular cholesterol homeostasis through the expression of the major cholesterol exporter ABCA1 with apoptotic cell removal and inflammatory responses.

CD109 released from human bone marrow mesenchymal stem cells attenuates TGF-β-induced epithelial to mesenchymal transition and stemness of squamous cell carcinoma.

Although there is increasing evidence that human bone marrow mesenchymal stem cells (hBM-MSCs) play an important role in cancer progression, the underlying mechanisms are poorly understood. Transforming growth factor β (TGF-β) is an important pro-metastatic cytokine. We have previously shown that CD109, a glycosylphosphatidylinositol-anchored protein, is a TGF-β co-receptor and a strong inhibitor of TGF-β signalling. Moreover, CD109 can be released from the cell surface. In the current study, we examined whether hBM-MSCs regulate the malignant properties of squamous cell carcinoma cells, and whether CD109 plays a role in mediating the effect of hBM-MSCs on cancer cells. Here we show that hBM-MSC-conditioned medium decreases proliferation and induces apoptosis in human squamous carcinoma cell lines, A431 and FaDu. Importantly, hBM-MSC-conditioned medium markedly suppresses markers of epithelial-to-mesenchymal transition and stemness, and concomitantly decreases cell migration, invasion, and spheroid formation in A431 and FaDu cells. In addition, knockdown of CD109 in hBM-MSCs abrogates the anti-malignant activity of hBM-MSC-conditioned medium on A431 and FaDu cells. Furthermore, overexpression of CD109 in A431 cells decreases their malignant traits. Together, our findings suggest that hBM-MSCs inhibit the malignant traits of squamous cell carcinoma cells by a paracrine effect via released factors and that CD109 released from hBM-MSCs, at least partially, mediates these effects.

Release of endothelial cell associated VEGFR2 during TGF-\u03b2 modulated angiogenesis in vitro

BackgroundSprouting angiogenesis requires vascular endothelial proliferation, migration and morphogenesis. The process is regulated by soluble factors, principally vascular endothelial growth factor (VEGF), and via bidirectional signaling through the Jagged/Notch system, leading to assignment of tip cell and stalk cell identity. The cytokine transforming growth factor beta (TGF-β) can either stimulate or inhibit angiogenesis via its differential surface receptor signaling. Here we evaluate changes in expression of angiogenic signaling receptors when bovine aortic endothelial cells were exposed to TGF-β1 under low serum conditions.ResultsTGF-β1 induced a dose dependent inhibition of tip cell assignment and subsequent angiogenesis on Matrigel, maximal at 5.0 ng/ml. This occurred via ALK5-dependent pathways and was accompanied by significant upregulation of the TGF-β co-receptor endoglin, and SMAD2 phosphorylation, but no alteration in Smad1/5 activation. TGF-β1 also induced ALK5-dependent downregulation of Notch1 but not of its ligand delta-like ligand 4. Cell associated VEGFR2 (but not VEGFR1) was significantly downregulated and accompanied by reciprocal upregulation of VEGFR2 in conditioned medium. Quantitative polymerase chain reaction analysis revealed that this soluble VEGFR2 was not generated by a selective shift in mRNA isoform transcription. This VEGFR2 in conditioned medium was full-length protein and was associated with increased soluble HSP-90, consistent with a possible shedding of microvesicles/exosomes.ConclusionsTaken together, our results suggest that endothelial cells exposed to TGF-β1 lose both tip and stalk cell identity, possibly mediated by loss of VEGFR2 signaling. The role of these events in physiological and pathological angiogenesis requires further investigation.

Altering the Proteoglycan State of Transforming Growth Factor β Type III Receptor (TβRIII)/Betaglycan Modulates Canonical Wnt/β-Catenin Signaling

Hyperactive Wnt/β-catenin signaling is linked to cancer progression and developmental abnormalities, making identification of mechanisms controlling Wnt/β-catenin signaling vital. Transforming growth factor β type III receptor (TβRIII/betaglycan) is a transmembrane proteoglycan co-receptor that exists with or without heparan and/or chondroitin sulfate glycosaminoglycan (GAG) modifications in cells and has established roles in development and cancer. Our studies here demonstrate that TβRIII, independent of its TGFβ co-receptor function, regulates canonical Wnt3a signaling by controlling Wnt3a availability through its sulfated GAG chains. Our findings revealed, for the first time, opposing functions for the different GAG modifications on TβRIII suggesting that Wnt interactions with the TβRIII heparan sulfate chains result in inhibition of Wnt signaling, likely via Wnt sequestration, whereas the chondroitin sulfate GAG chains on TβRIII promote Wnt3a signaling. These studies identify a novel, dual role for TβRIII/betaglycan and define a key requirement for the balance between chondroitin sulfate and heparan sulfate chains in dictating ligand responses with implications for both development and cancer.

Overexpression of CD109 in the Epidermis Differentially Regulates ALK1 Versus ALK5 Signaling and Modulates Extracellular Matrix Synthesis in the Skin

Transforming growth factor-β (TGF-β) is a multifunctional growth factor involved in many physiological processes including wound healing and inflammation. Excessive TGF-β signaling in the skin has been implicated in fibrotic skin disorders such as keloids and scleroderma. We previously identified CD109 as a TGF-β co-receptor and inhibitor of TGF-β signaling and have shown that transgenic mice overexpressing CD109 in the epidermis display decreased scarring. In certain cell types, in addition to the canonical type I receptor, ALK5, which activates Smad2/3, TGF-β can signal through another type I receptor, ALK1, which activates Smad1/5. Here we demonstrate that ALK1 is expressed and co-localizes with CD109 in mouse keratinocytes and that mice overexpressing CD109 in the epidermis display enhanced ALK1-Smad1/5 signaling but decreased ALK5-Smad2/3 signaling, TGF-β expression, and extracellular matrix production in the skin when compared with wild-type littermates. Furthermore, treatment with conditioned media from isolated keratinocytes or epidermal explants from CD109 transgenic mouse skin leads to a decrease in extracellular matrix production in mouse skin fibroblasts. Taken together, our findings suggest that CD109 differentially regulates TGF-β-induced ALK1-Smad1/5 versus ALK5-Smad2/3 pathways, leading to decreased extracellular matrix production in the skin and that epidermal CD109 expression regulates dermal function through a paracrine mechanism.

Abstract 941: Differential roles for membrane-bound and soluble CD109 in breast cancer progression

Abstract Introduction: Our group has recently reported the identification of CD109 (a GPI anchored cell surface glycoprotein) as a TGF-β co-receptor and potent antagonist of TGF-β signaling and responses in human epithelial cells in vitro and in vivo. Proteinase mediated shedding converts CD109 from a membrane-bound coreceptor into a soluble effector capable of binding TGF-β. Our results indicate that CD109 overexpression inhibits TGF-β -induced Epithelial Mesenchymal Transition (EMT) and migration in breast cancer cells in vitro. However, in breast carcinomas, CD109 overexpression correlates with poor prognosis and an aggressive phenotype. Our findings together with the recent report of over expression of CD109 in breast cancer provide compelling reasons to examine whether membrane anchored or soluble CD109 plays an essential role in breast cancer progression and thus represent a molecular target for the treatment of breast cancer. Methods: To investigate the role of CD109 in breast cancer cells that overexpress membrane-anchored (m)CD109 or soluble (s)CD109, or in which endogenous CD109 expression is blocked, or breast cancer cells treated with recombinant CD109 protein, were analysed for SMAD signaling and EMT markers (Fibronectin, PAI, Vimentin, N-cadherin, α-sma, Snail) using immunoblotting. Cell migration and invasion potential in response to TGF-β were assessed using scratch assay and boyden chamber assay, respectively. Results: To determine the role of CD109 in breast cancer progression, human MDA-MB-231 breast cancer cells were transfected with plasmid overexpressing wild-type (WT) which has the potential to shed CD109. Overexpression of WT CD109 decreased TGF-β induced phospho-SMAD signaling, EMT, migration and invasion of breast cancer cells. Conversely, treatment of Hs578T breast cancer cells with CD109 siRNA increased TGF-β induced phospho SMAD signaling, EMT, migration and invasion of cancer cells. In an alternate approach breast cancer cells treated with recombinant CD109 (resembling the shed form of CD109) also showed decreased TGF-β induced EMT, migration and invasion of cancer cells. However overexpression of membrane bound (furinase-mediated shedding resistant) form of CD109 in MDA-MB-231 breast cancer cells showed increased EMT, migration and invasion of cancer cells. Analysis of downstream signaling showed that although both membrane bound and soluble form of CD109 attenuates TGF-β signaling, the membrane bound CD109 promotes tumor aggressiveness. Conclusion: Taken together, these results suggest that membrane anchored and soluble form of CD109 have different role in breast cancer. Both membrane bound and soluble CD109 inhibit TGF-β signaling in breast cancer however the membrane bound form of CD109 can promote cancer progression. Mechanisms underlying the differential effect of these two forms of CD109 in breast cancer need to be deciphered. Citation Format: Priyanka Sehgal, Anie Philip. Differential roles for membrane-bound and soluble CD109 in breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 941.

Abstract B15: Transforming growth factor β type III (TβRIII/Betaglycan) suppresses canonical Wnt signaling in ovarian cancer

Abstract Ovarian cancer remains the deadliest gynecologic malignancy and the fifth leading cause of death from cancer in women[1]. Transforming growth factor β Type III (TβRIII/Betaglycan) receptor is a ubiquitously expressed co-receptor for the TGF-β ligand superfamily[2] whose expression is significantly decreased or lost in human epithelial ovarian tumors compared to normal tissue, with loss of TβRIII expression correlating with tumor grade and ovarian cancer progression[3]. TβRIII has increasing roles in suppressing cancer progression via cell motility[4], cell adhesion[5], and cell differentiation[2], largely independent of its TGF-β co-receptor functions[2, 3]. Structurally, TβRIII is a transmembrane proteoglycan containing both heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chain modifications in its extracellular domain[5] that can interact with non-TGF-β family members, namely FGF2, via its GAG chains to regulate tumorigenesis[2].There is increasing evidence that Wnt glycoproteins that play complex roles in cancer[1, 6-11] have a high affinity for GAG chains on proteoglycans[12, 13]. Thus, we have initiated studies to determine the possible role of TβRIII on canonical Wnt3a signaling in ovarian cancer. Using a combination of cell signaling and biochemical approaches in ovarian cancer models, we find that TβRIII suppresses Wnt3a signaling by binding Wnt3a via its heparan sulfated GAG chains, which leads to perturbation of the LRP6/Frizzled/Wnt3a complex required for Wnt3a signaling activation. Reciprocal overexpression and knockdown of TβRIII in ovarian cancer cell lines reveal that TβRIII is a strong suppressor of canonical Wnt3a signaling, largely independent of TβRIII’s role as a TGF-β co-receptor. Finally, we provide evidence, via TCF/LEF-sensitive reporter activity[14], that TβRIII chondroitin sulfate chains can promote Wnt3a signaling in the absence of its heparan sulfate chains. These results indicate an intricate mode of Wnt3a signaling regulation by TβRIII/Betaglycan, mediated by distinct functions of its heparan and chondroitin chains. Our studies suggest a critical role for TβRIII’s heparan and chondroitin sulfated GAG modifications in modulating Wnt3a signaling and, more importantly, a possible therapeutic approach to address malfunctions in the Wnt signaling pathways that lead to ovarian carcinogenesis. Citations: 1. Arend, R.C., et al., The Wnt/beta-catenin pathway in ovarian cancer: a review. Gynecol Oncol, 2013. 131(3): p. 772-9. 2. Knelson, E.H., et al., Type III TGF-beta receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma. J Clin Invest, 2013. 123(11): p. 4786-98. 3. Gatza, C.E., S.Y. Oh, and G.C. Blobe, Roles for the type III TGF-beta receptor in human cancer. Cell Signal, 2010. 22(8): p. 1163-74. 4. Mythreye, K., et al., TbetaRIII/beta-arrestin2 regulates integrin alpha5beta1 trafficking, function, and localization in epithelial cells. Oncogene, 2013. 32(11): p. 1416-27. 5. Mythreye, K. and G.C. Blobe, Proteoglycan signaling co-receptors: roles in cell adhesion, migration and invasion. Cell Signal, 2009. 21(11): p. 1548-58. 6. Tung, E.K., et al., Upregulation of the Wnt co-receptor LRP6 promotes hepatocarcinogenesis and enhances cell invasion. PLoS One, 2012. 7(5): p. e36565. 7. Qi, L., et al., Wnt3a expression is associated with epithelial-mesenchymal transition and promotes colon cancer progression. J Exp Clin Cancer Res, 2014. 33(1): p. 107. 8. Lavergne, E., et al., Blocking Wnt signaling by SFRP-like molecules inhibits in vivo cell proliferation and tumor growth in cells carrying active beta-catenin. Oncogene, 2011. 30(4): p. 423-33. 9. Verras, M., et al., Wnt3a growth factor induces androgen receptor-mediated transcription and enhances cell growth in human prostate cancer cells. Cancer Res, 2004. 64(24): p. 8860-6. 10. Gatcliffe, T.A., et al., Wnt signaling in ovarian tumorigenesis. Int J Gynecol Cancer, 2008. 18(5): p. 954-62. 11. Boyer, A., A.K. Goff, and D. Boerboom, WNT signaling in ovarian follicle biology and tumorigenesis. Trends Endocrinol Metab, 2010. 21(1): p. 25-32. 12. Ai, X., et al., QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling. J Cell Biol, 2003. 162(2): p. 341-51. 13. Capurro, M., et al., Glypican-3 binds to Frizzled and plays a direct role in the stimulation of canonical Wnt signaling. J Cell Sci, 2014. 127(Pt 7): p. 1565-75. 14. Veeman, M.T., et al., Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr Biol, 2003. 13(8): p. 680-5. Citation Format: Laura M. Jenkins, Archana Varadaraj, Haley Flores, Mythreye Karthikeyan. Transforming growth factor β type III (TβRIII/Betaglycan) suppresses canonical Wnt signaling in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B15.