Dianhua Qiao

Shedding of syndecan-1 by stromal fibroblasts stimulates human breast cancer cell proliferation via FGF2 activation.

The cell surface heparan sulfate proteoglycan syndecan-1 is induced in stromal fibroblasts of breast carcinomas and participates in a reciprocal feedback loop, which stimulates carcinoma cell growth in vitro and in vivo. To define the molecular mechanism of carcinoma growth stimulation, a three-dimensional co-culture model was developed that combines T47D breast carcinoma cells with immortalized human mammary fibroblasts in collagen gels. By silencing endogenous syndecan-1 induction with short interfering RNA and expressing mutant murine syndecan-1 constructs, it was determined that carcinoma cell mitogenesis required proteolytic shedding of syndecan-1 from the fibroblast surface. The paracrine growth signal was mediated by the syndecan-1 heparan lfate chains rather than the ectodomain of the core protein and required fibroblast growth factor 2 and stroma-derived factor 1. This paracrine pathway may provide an opportunity for the therapeutic disruption of stromaepithelial signaling.

Membrane Type 1 Matrix Metalloproteinase–Mediated Stromal Syndecan-1 Shedding Stimulates Breast Carcinoma Cell Proliferation

Mounting evidence implicates stromal fibroblasts in breast carcinoma progression. We have recently shown in three-dimensional coculture experiments that human mammary fibroblasts stimulate the proliferation of T47D breast carcinoma cells and that this activity requires the shedding of the heparan sulfate proteoglycan syndecan-1 (Sdc1) from the fibroblast surface. The goal of this project was to determine the mechanism of Sdc1 ectodomain shedding. The broad spectrum matrix metalloproteinase (MMP) inhibitor GM6001 specifically blocked Sdc1-mediated carcinoma cell growth stimulation, pointing toward MMPs as critical enzymes involved in Sdc1 shedding. MMP-2 and membrane type 1 MMP (MT1-MMP) were the predominant MMPs expressed by the mammary fibroblasts. Fibroblast-dependent carcinoma cell growth stimulation in three-dimensional coculture was abolished by MT1-MMP expression silencing with small interfering RNA and restored either by adding recombinant MT1-MMP catalytic domain or by expressing a secreted form of Sdc1 in the fibroblasts. These findings are consistent with a model where fibroblast-derived MT1-MMP cleaves Sdc1 at the fibroblast surface, leading to paracrine growth stimulation of carcinoma cells by Sdc1 ectodomain. The relevance of MT1-MMP in paracrine interactions was further supported by coculture experiments with T47D cells and primary fibroblasts isolated from human breast carcinomas or matched normal breast tissue. Carcinoma-associated fibroblasts stimulated T47D cell proliferation significantly more than normal fibroblasts in three-dimensional coculture. Function-blocking anti-MT1-MMP antibody significantly inhibited the T47D cell growth stimulation in coculture with primary fibroblasts. In summary, these results ascribe a novel role to fibroblast-derived MT1-MMP in stromal-epithelial signaling in breast carcinomas.

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.

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.

Retraction for Qiao et al., Glypican 1 Stimulates S Phase Entry and DNA Replication in Human Glioma Cells and Normal Astrocytes

A replication-deficient adenovirus (pLP-Adeno-X-PRLS; Clontech) was used to overexpress glypican 1 (GPC1), and empty adenovirus (Ad-control) was used as control. Overexpression of GPC1 was confirmed by Western blotting. Recently, we performed transmission electron microscopy and detected adenoviral particles in Ad-GPC1-infected but not Ad-control-infected cells. PCR analysis confirmed the presence of E1 protein in Ad-GPC1 but not in Ad-control. Therefore, we conclude that Ad-GPC1 has acquired the E1 gene from the HEK 293 packaging cells and has become replication competent. Considering the small scale of virus production used in these experiments, this is a very unlikely event (H. Lochmüller et al., Hum Gene Ther, 5:1485–1491, 1994; J. Zhu et al., Hum Gene Ther, 10:113–121, 1999), which appears to have occurred.

Abstract 1307: c-Myc is a key mediator of glypican-1 (GPC1)-dependent deregulation of the cell cycle

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Human gliomas show extraordinary cellular and genomic heterogeneity while the heparan sulfate proteoglycan glypican-1 (GPC1) is nearly universally overexpressed in human gliomas but absent in normal glia. Cell membrane-associated heparan sulfate proteoglycans, including GPC1, can enhance the activity of many heparan sulfate-binding growth factors and cytokines to promote cell growth and development. Our previous studies using normal human astrocytes and U87-MG, a human glioma cell line carrying relatively low basal levels of GPC1, showed that transduction of GPC1 dramatically stimulates G1-S cell cycle progression and DNA replication, ultimately leading to DNA re-replication and DNA damage. This cell cycle effect of GPC1 appears to be at least partially attributable to the deregulation of G1/S checkpoint resulting from significant downregulation of pRb, p21Cip/p27Kip and CDH1 (FZR1) and significant upregulation of cyclin E, cyclin-dependent kinase 2 (CDK2) and Skp2 by GPC1 {Qiao, D., et al., Mol. Cell. Biol. 2013, 33(22):4408}. Here we further report that c-Myc acts as a key mediator for GPC1-mediated G1-S cell cycle deregulation. Both c-Myc protein and transactivation activity can be significantly induced by ectopic expression of GPC1 in a cell type-independent fashion. Inhibition of GPC1-induced c-Myc transactivation by the c-Myc/Max-specific inhibitor 10058-F4 almost completely abolishes GPC1-induced E2F activation and DNA re-replication. Consistent with these results, inhibition of Myc activity completely restores the protein levels of cyclin E, CDK2, pRb, p27Kip and Skp2 which were significantly altered by expression of GPC1. GPC1-induced reduction of p21Cip was partially restored upon inhibition of c-Myc. Expression of GPC1 also induced cell detachment and rounding, accompanied by a significant downregulation of the β1 and β3 integrin subunits. Inhibition of Myc effectively blocked GPC1 induction of both cell detachment/rounding and downregulation of β1 and β3 integrins. These results suggest a key role for c-Myc in GPC-1-mediated cellular effects and cell signaling. In addition to direct transcriptional activation of cyclin E, E2Fs and Skp2, c-Myc may also modulate specific protein degradation activities. The c-Myc-mediated transcriptional repression of p21Cip/p27Kip and of integrins may also contribute to GPC1-induced effects on the cell cycle and on cell morphology. Given the potent oncogenic activity of c-Myc and the fact that c-Myc is required for the maintenance of stemness of glioma stem cells, our data imply that GPC1 may play a significant role in both glioma tumorigenesis and growth. Further in vitro and in vivo studies are required to fully understand the role and mechanisms of both GPC1 and c-Myc in human gliomas. Citation Format: Dianhua Qiao, Kristy Meyer, Andreas Friedl. c-Myc is a key mediator of glypican-1 (GPC1)-dependent deregulation of the cell cycle. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1307. doi:10.1158/1538-7445.AM2014-1307

Abstract 3042: Glypican-1 (GPC1) promotes S-phase entry and DNA replication in human glioma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Glypican-1 (GPC1), the most ubiquitously expressed member of the glypican family of heparan sulfate proteoglycans (HSPGs), is highly expressed in the developing brain and gliomas. In gliomas, GPC1 is overexpressed in both tumor blood vessel endothelial cells (ECs) and the neoplastic cells. Prior work by us in ECs indicates that GPC1 is overall mitogenic and essential for cell cycle progression and proliferation, implying a role in glioma angiogenesis. The present study focused on the role of GPC1 in glioma cells to explore potential roles in tumorigenesis and growth. The expression of GPC1 in U87 cells, a human glioma cell line with a relatively low basal level of GPC1, was titrated by adenoviral transduction. BrdU pulse-labeling showed a robust induction of S-phase entry and DNA replication by expression of GPC1 in a dose-dependent manner. GPC1 overexpression ultimately led to DNA re-replication and aneuploidy as well as enlarged nuclei and cell rounding. Consistent with these cell cycle effects, ectopic expression of GPC1 significantly induced downregulation of pRb, p21Cip1 and p27Kip1 and upregulation of cyclin E, CDK2 and E2F transactivation activity. These cellular and molecular activities of GPC1 were independent of serum concentrations in the growth medium, suggesting that they do not require exogenous growth factors. This activity required intact GPC1 as neither unglycanated GPC1 core protein nor isolated GPC1 HS chains induced the cell cycle and molecular alterations seen after intact GPC1 was added. The more general relevance of our observation to other cell types and the HS-dependency of the GPC1 activity were confirmed by GPC1 transduction of wild-type and HS synthesis-defective CHO cells. Overexpression of GPC1 induced dramatic S-phase entry in wild-type but not in HS-deficient mutant CHO cells. In summary, this work reveals a potent S-phase promoting activity of GPC1 in glioma cells, which may lead to accelerated cell growth and/or increased genomic instability and, therefore, implies a role for GPC1 in glioma tumorigenesis and growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3042. doi:1538-7445.AM2012-3042