Kristy Meyer

HSulf-1 inhibits angiogenesis and tumorigenesis in vivo.

We previously identified HSulf-1 as a down-regulated gene in several tumor types including ovarian, breast, and hepatocellular carcinomas. Loss of HSulf-1, which selectively removes 6-O-sulfate from heparan sulfate, up-regulates heparin-binding growth factor signaling and confers resistance to chemotherapy-induced apoptosis. Here we report that HSulf-1 expression in MDA-MB-468 breast carcinoma clonal lines leads to reduced proliferation in vitro and reduced tumor burden in athymic nude mice in vivo. Additionally, xenografts derived from HSulf-1-expressing stable clones of carcinoma cells showed reduced vessel density, marked necrosis, and apoptosis, indicative of inhibition of angiogenesis. Consistent with this observation, HSulf-1-expressing clonal lines showed reduced staining with the endothelial marker CD31 in Matrigel plug assay, indicating that HSulf-1 expression inhibits angiogenesis. More importantly, HSulf-1 expression in the xenografts was associated with a reduced ability of vascular endothelial cell heparan sulfate to participate in a complex with fibroblast growth factor 2 (FGF-2) and its receptor tyrosine kinase FGF receptor 1c. In vitro, short hairpin RNA-mediated down-regulation of HSulf-1 in human umbilical vein endothelial cells (HUVEC) resulted in an increased proliferation mediated by heparan sulfate-dependent FGF-2, hepatocyte growth factor, and vascular endothelial growth factor 165 (VEGF165) but not by heparan sulfate-independent VEGF121. HSulf-1 down-regulation also enhanced downstream signaling through the extracellular signal-regulated kinase pathway compared with untreated cells. Consistent with the role of heparan sulfate glycosaminoglycan sulfation in VEGF-mediated signaling, treatment of HUVEC cells with chlorate, which inhibits heparan sulfate glycosaminoglycan sulfation and therefore mimics HSulf-1 overexpression, led to an attenuated VEGF-mediated signaling. Collectively, these observations provide the first evidence of a novel mechanism by which HSulf-1 modulates the function of heparan sulfate binding VEGF165 in proliferation and angiogenesis.

Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 receptor binding in breast carcinomas.

Binding of fibroblast growth factors (FGFs) to their tyrosine kinase-signaling receptors (FGFRs) requires heparan sulfate (HS). HS proteoglycans (HSPGs) determine mitogenic responses of breast carcinoma cells to FGF-2 in vitro. For this study, we examined the role of HSPGs as modulators of FGF-2 binding to FGFR-1 in situ and in vitro. During stepwise reconstitution of the FGF-2/HSPG/FGFR-1 complex in situ, we identified an elevated ability of breast carcinoma cell HSPGs to promote receptor complex formation compared to normal breast epithelium. HSPGs isolated from the MCF-7 breast-carcinoma cell line were then fractionated according to their ability to assemble the FGF-2 receptor complex. All MCF-7 HSPGs are decorated with HS chains similarly capable of promoting FGF-2 receptor complex formation. In this in vitro model, syndecan-1 and syndecan-4 are the cell surface HSPGs contributing most to the complex formation. Relative expression levels of these syndecans in human breast carcinoma tissues correlate well with receptor complex formation in situ, indicating that in breast carcinomas, core protein levels determine FGF-2 receptor complex formation. However, variances in syndecan expression levels do not explain the difference in FGF-2 receptor complex formation between normal and malignant epithelial cells, suggesting that alterations in HS structure occur during malignant transformation.

Differential ability of heparan sulfate proteoglycans to assemble the fibroblast growth factor receptor complex in situ

Fibroblast growth factors (FGFs) require heparan sulfate proteoglycans (HSPGs) as cofactors for signaling. The heparan sulfate chains (HS) mediate stable high affinity binding of FGFs to their receptor tyrosine kinases (FR) and may specifically regulate FGF activity. A novel in situ binding assay was developed to examine the ability of HSPGs to promote FGF/FR binding using a soluble FR fusion construct (FR1‐AP). This fusion protein probe forms a dimer in solution, simulating the dimerization or oligomerization that is thought to occur at the cell surface physiologically. In frozen sections of human skin, FGF‐2 binds to keratinocytes and basement membranes of epidermis and dermal blood vessels. In contrast, in skin preincubated with FGF‐2, FR1‐AP binds avidly to FGF‐2 immobilized on keratinocyte cell surfaces, but fails to bind to basement membranes at the dermo‐epidermal junction or dermal microvessels despite the fact that these structures bind large amounts of FGF‐2. Apparently, basement membrane and cell surface HSPGs differ in their ability to mediate the assembly of a FGF/FR signaling complex presumably due to structural differences of the heparan sulfate chains.—Chang, Z., Meyer, K., Rapraeger, A. C., Friedl, A. Differential ability of heparan sulfate proteoglycans to assemble the fibroblast growth factor receptor complex in situ. FASEB J. 14, 137–144(2000)

Signal Transducers and Activators of Transcription Mediate Fibroblast Growth Factor–Induced Vascular Endothelial Morphogenesis

The fibroblast growth factors (FGF) play diverse roles in development, wound healing, and angiogenesis. The intracellular signal transduction pathways, which mediate these pleiotropic activities, remain incompletely understood. We show here that the proangiogenic factors FGF2 and FGF8b can activate signal transducers and activators of transcription (STAT) in mouse microvascular endothelial cells (EC). Both FGF2 and FGF8b activate STAT5 and to a lesser extent STAT1, but not STAT3. The FGF2-dependent activation of endothelial STAT5 was confirmed in vivo with the Matrigel plug angiogenesis assay. In tissue samples of human gliomas, a tumor type wherein FGF-induced angiogenesis is important, STAT5 is detected in tumor vessel EC nuclei, consistent with STAT5 activation. By forced expression of constitutively active or dominant-negative mutant STAT5A in mouse brain ECs, we further show that STAT5 activation is both necessary and sufficient for FGF-induced cell migration, invasion, and tube formation, which are key events in vascular endothelial morphogenesis and angiogenesis. In contrast, STAT5 is not required for brain EC mitogenesis. The cytoplasmic tyrosine kinases Src and Janus kinase 2 (Jak2) both seem to be involved in the activation of STAT5, as their inhibition reduces FGF2- and FGF8b-induced STAT5 phosphorylation and EC tube formation. Constitutively active STAT5A partially restores tube formation in the presence of Src or Jak2 inhibitors. These observations show that FGFs use distinct signaling pathways to induce angiogenic phenotypes. Together, our findings implicate the FGF-Jak2/Src-STAT5 cascade as a critical angiogenic FGF signaling pathway.

STAT5 and Prolactin Participate in a Positive Autocrine Feedback Loop That Promotes Angiogenesis

Background: Active STAT5 promotes angiogenesis. Results: In human brain endothelial cells, active STAT5 promotes the secretion of prolactin, which stimulates endothelial cell migration and tube formation. Prolactin also induces the secretion of VEGF and activates STAT5. Conclusion: Prolactin and STAT5 are engaged in a positive feedback loop that stimulates angiogenesis. Significance: Prolactin may be important in pathologic angiogenesis. We have shown previously that the murine prolactin/growth hormone family member proliferin plays a pivotal role in angiogenesis induced by the FGF2/STAT5 signaling cascade. To delineate the signaling pathway downstream of STAT5 in the human system, where proliferin does not exist, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in hCMEC/D3 human brain endothelial cells. We found that conditioned medium from CA-STAT5A- but not from DN-STAT5A-overexpressing endothelial cells (EC) is sufficient to induce EC migration and tube formation but not proliferation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. We identified prolactin (PRL) as a candidate autocrine factor. CA-STAT5A expression stimulates PRL production at the RNA and protein level, and STAT5A binds to the PRL promoter region, suggesting direct transcriptional regulation. Medium conditioned by CA-STAT5A-overexpressing EC induces phosphorylation of the PRL receptor and activates MAPK. Knockdown of PRL expression by shRNA or blocking of PRL activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. The addition of recombinant PRL restores this activity. STAT5A-induced PRL in the conditioned medium can activate STAT5, STAT1, and to a lesser extent STAT3 in hCMEC/D3 cells, suggesting the existence of a positive feedback loop between STAT5 and PRL that promotes angiogenesis. Furthermore, we find that VEGF, a potent proangiogenic factor, is induced by activation of STAT5A, and VEGF induction depends on PRL expression. These observations demonstrate a STAT5/PRL/VEGF signaling cascade in human brain EC and implicate PRL and VEGF as autocrine regulators of EC migration, invasion, and tube formation.

Glypican-1 Stimulates Skp2 Autoinduction Loop and G1/S Transition in Endothelial Cells

Background: Glypican-1 is a cell surface heparan sulfate proteoglycan that regulates cell growth. Results: In endothelial cells, glypican-1 regulates a variety of cell cycle effectors leading to increased S phase entry. Conclusion: Glypican-1 inactivates the G1/S checkpoint, apparently by activating the Skp2 autoinduction loop. Significance: These findings provide mechanistic insights into how glypican-1 regulates the cell cycle and proliferation. The heparan sulfate proteoglycan glypican-1 (GPC1) is involved in tumorigenesis and angiogenesis and is overexpressed frequently in tumor and endothelial cells (ECs) in human gliomas. We demonstrated previously that in brain EC, GPC1 regulates mitotic cyclins and securin as well as mitosis and that GPC1 is required for progression through the cell cycle. To characterize the molecular mechanism underlying cell cycle regulation by GPC1, we systematically investigated its effects on key G1/S checkpoint regulators and on major signaling pathways reportedly activated by Dally (Division abnormally delayed) the Drosophila GPC1 homologue. We found that elevated GPC1 affected a wide range of G1/S checkpoint regulators, leading to inactivation of the G1/S checkpoint and increased S phase entry, apparently by activating the mitogen-independent Skp2 autoinduction loop. Specifically, GPC1 suppressed CDK inhibitors (CKIs), including p21, p27, p16, and p19, and the D cyclins, and induced CDK2 and Skp2. GPC1 may trigger the Skp2 autoinduction loop at least partially by suppressing p21 transcription as knockdown of p21 by RNAi can mimic the effect of GPC1 on the cell cycle regulators related to the loop. Moreover, multiple mitogenic signaling pathways, including ERK MAPK, Wnt and BMP signaling, were significantly stimulated by GPC1 as has been reported for Dally in Drosophila. Notably, the c-Myc oncoprotein, which is frequently up-regulated by both ERK and Wnt signaling and functions as a potent transcription repressor for CKIs as well as D cyclins, was also significantly induced by GPC1. These findings provide mechanistic insights into how GPC1 regulates the cell cycle and proliferation.

Glypican-1 Regulates Anaphase Promoting Complex/Cyclosome Substrates and Cell Cycle Progression in Endothelial Cells

Glypican-1 (GPC1), a member of the mammalian glypican family of heparan sulfate proteoglycans, is highly expressed in glioma blood vessel endothelial cells (ECs). In this study, we investigated the role of GPC1 in EC replication by manipulating GPC1 expression in cultured mouse brain ECs. Moderate GPC1 overexpression stimulates EC growth, but proliferation is significantly suppressed when GPC1 expression is either knocked down or the molecule is highly overexpressed. Flow cytometric and biochemical analyses show that high or low expression of GPC1 causes cell cycle arrest at mitosis or the G2 phase of the cell cycle, accompanied by endoreduplication and consequently polyploidization. We further show that GPC1 inhibits the anaphase-promoting complex/cyclosome (APC/C)-mediated degradation of mitotic cyclins and securin. High levels of GPC1 induce metaphase arrest and centrosome overproduction, alterations that are mimicked by overexpression of cyclin B1 and cyclin A, respectively. These observations suggest that GPC1 regulates EC cell cycle progression at least partially by modulating APC/C-mediated degradation of mitotic cyclins and securin.

Angiogenesis induced by signal transducer and activator of transcription 5A (STAT5A) is dependent on autocrine activity of proliferin

Background: FGFs activate STAT5 transcription factor. Results: In mouse endothelial cells, active STAT5 promotes the production and release of proliferin, which stimulates endothelial cell migration, invasion and tube formation in vitro, and angiogenesis in vivo. Conclusion: Proliferin is a secreted pro-angiogenic factor downstream of FGFs and STAT5. Significance: Proliferin is an autocrine factor likely relevant in physiologic and pathologic angiogenesis. Multiple secreted factors induce the formation of new blood vessels (angiogenesis). The signal transduction events that orchestrate the numerous cellular activities required for angiogenesis remain incompletely understood. We have shown previously that STAT5 plays a pivotal role in angiogenesis induced by FGF2 and FGF8b. To delineate the signaling pathway downstream of STAT5, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in mouse brain endothelial cells (EC). We found that the conditioned medium from CA-STAT5A but not from dominant-negative STAT5A overexpressing EC is sufficient to induce EC invasion and tube formation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. Conversely, CA-STAT5A-induced conditioned medium had no effect on EC proliferation. Using a comparative genome-wide transcription array screen, we identified the prolactin family member proliferin (PLF1 and PLF4) as a candidate autocrine factor. The CA-STAT5A-dependent transcription and secretion of PLF by EC was confirmed by quantitative RT-PCR and Western blotting, respectively. CA-STAT5A binds to the PLF1 promoter region, suggesting a direct transcriptional regulation. Knockdown of PLF expression by shRNA or by blocking of PLF activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. Similarly, the ability of concentrated conditioned medium from CA-STAT5A transfected EC to induce angiogenesis in Matrigel plugs in vivo was abolished when PLF was depleted from the medium. These observations demonstrate a FGF/STAT5/PLF signaling cascade in EC and implicate PLF as autocrine regulator of EC invasion and tube formation.

Glypican 1 Stimulates S Phase Entry and DNA Replication in Human Glioma Cells and Normal Astrocytes

ABSTRACT Malignant gliomas are highly lethal neoplasms with limited treatment options. We previously found that the heparan sulfate proteoglycan glypican 1 (GPC1) is universally and highly expressed in human gliomas. In this study, we investigated the biological activity of GPC1 expression in both human glioma cells and normal astrocytes in vitro. Expression of GPC1 inactivates the G1/S checkpoint and strongly stimulates DNA replication. Constitutive expression of GPC1 causes DNA rereplication and DNA damage, suggesting a mutagenic activity for GPC1. GPC1 expression leads to a significant downregulation of the tumor suppressors pRb, Cip/Kip cyclin-dependent kinase inhibitors (CKIs), and CDH1, and upregulation of the pro-oncogenic proteins cyclin E, cyclin-dependent kinase 2 (CDK2), Skp2, and Cdt1. These GPC1-induced changes are accompanied by a significant reduction in all types of D cyclins, which is independent of serum supplementation. It is likely that GPC1 stimulates the so-called Skp2 autoinduction loop, independent of cyclin D-CDK4/6. Knockdown of Skp2, CDK2, or cyclin E, three key elements within the network modulated by GPC1, results in a reduction of the S phase and aneuploid fractions, implying a functional role for these regulators in GPC1-induced S phase entry and DNA rereplication. In addition, a significant activation of both the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways by GPC1 is seen in normal human astrocytes even in the presence of growth factor supplement. Both pathways are constitutively activated in human gliomas. The surprising magnitude and the mitogenic and mutagenic nature of the effect exerted by GPC1 on the cell cycle imply that GPC1 may play an important role in both glioma tumorigenesis and growth.

Thyroid cancer stem-like cell exosomes: regulation of EMT via transfer of lncRNAs

Thyroid cancers are the most common endocrine malignancy and approximately 2% of thyroid cancers are anaplastic thyroid carcinoma (ATC), one of the most lethal and treatment resistant human cancers. Cancer stem-like cells (CSCs) may initiate tumorigenesis, induce resistance to chemotherapy and radiation therapy, have multipotent capability and may be responsible for recurrent and metastatic disease. The production of CSCs has been linked to epithelial-mesenchymal transition (EMT) and the acquisition of stemness. Exosomes are small (30–150 nm) membranous vesicles secreted by most cells that play a significant role in cell-to-cell communication. Many non-coding RNAs (ncRNA), such as long-non-coding RNAs (lncRNA), can initiate tumorigenesis and the EMT process. Exosomes carry ncRNAs to local and distant cell populations. This study examines secreted exosomes from two in vitro cell culture models; an EMT model and a CSC model. The EMT was induced in a papillary thyroid carcinoma (PTC) cell line by TGFβ1 treatment. Exosomes from this model were isolated and cultured with naïve PTC cells and examined for EMT induction. In the CSC model, exosomes were isolated from a CSC clonal line, cultured with a normal thyroid cell line and examined for EMT induction. The EMT exosomes transferred the lncRNA MALAT1 and EMT effectors SLUG and SOX2; however, EMT was not induced in this model. The exosomes from the CSC model also transferred the lncRNA MALAT1 and the transcription factors SLUG and SOX2 but additionally transferred linc-ROR and induced EMT in the normal thyroid cells. Preliminary siRNA studies directed towards linc-ROR reduced invasion. We hypothesize that CSC exosomes transfer lncRNAs, importantly linc-ROR, to induce EMT and inculcate the local tumor microenvironment and the distant metastatic niche. Therapies directed towards CSCs, their exosomes and/or the lncRNAs they carry may reduce a tumor’s metastatic capacity.This report examined secreted exosomes from two in vitro cell culture models: an epithelial to mesenchymal transition model and a cancer stem-like cell model. The authors evaluated the expression and transfer of four long non-coding RNAs (HOTAIR, MALAT, PVT1, and linc-ROR) from exosomes derived from the models. They show that there is induced proliferation and invasive properties via the transfer of lncRNAs by exosomes.