Overexpression Of Glypican-1 Research Articles

Glypican-1 Overexpression in Different Types of Breast Cancers.

PurposeTreatment of metastatic breast cancer patients is challenging and remains a major underlying cause of female mortality. Understanding molecular alterations in tumor development is critical to identify novel biomarkers and targets for cancer diagnosis and therapy. One of the aberrant cancer expressions gaining recent research interest is glypican-1. Several studies reported strong glypican-1 expression in various types of human cancers. However, none of these investigated glypican-1 expression in a large cohort of breast cancer histopathological subtypes.Patients and MethodsImmunohistochemistry was used to assess glypican-1 expression in 220 breast cancer patients and its relation to demographic and clinical features, as well as important prognostic immunohistochemical markers for breast cancer.ResultsIntense glypican-1 expression was recognized in all breast cancer histopathological subtypes. Normal, healthy breast tissue displayed a heterogeneous low expression (20%). Importantly, a strong differential in glypican-1 expression was determined between normal and malignant breast tissues. Moreover, there was a significantly high rate of glypican-1 expression in advanced grades of breast cancer patients and larger tumor sizes. Unfortunately, the glypican-1 expression demonstrated no obvious relationship with the expression of various biomarkers in breast cancer.ConclusionThis study may establish glypican-1 as a promising new therapeutic target for the development of therapy in breast cancer.

Overexpression of Annexin A2 promotes proliferation by forming a Glypican 1/c-Myc positive feedback loop: prognostic significance in human glioma

In order to set up a reliable prediction system for the tumor grade and prognosis in glioma patients, we clarify the complicated crosstalk of Annexin A2 (ANXA2) with Glypican 1 (GPC1) and demonstrate whether combined indexes of ANXA2 and GPC1 could improve the prognostic evaluation for glioma patients. We found that ANXA2-induced glioma cell proliferation in a c-Myc-dependent manner. ANXA2 increased the expression of GPC1 via c-Myc and the upregulated GPC1 further promoted the c-Myc level, forming a positive feedback loop, which eventually led to enhanced proliferation of glioma cells. Both mRNA and protein levels of ANXA2 were upregulated in glioma tissues and coincided with the overexpression of GPC1. Besides, we utilized tissue microarrays (TMAs) and immunohistochemistry to demonstrate that glioma patients with both high expression of ANXA2 and GPC1 tended to have higher rate of tumor recurrence and shorter overall survival (OS). In conclusion, the overexpression of ANXA2 promotes proliferation of glioma cells by forming a GPC1/c-Myc positive feedback loop, and ANXA2 together with its downstream target GPC1 could be a potential “combination biomarker” for predicting prognosis of glioma patients.

Glypican-1, -3, -5 (GPC1, GPC3, GPC5) and Hedgehog Pathway Expression in Oral Squamous Cell Carcinoma.

Proteoglycans are involved in tumor development and may regulate the Hedgehog (HH) pathway. This study aimed to investigate the gene and protein expression of glypican-1 (GPC1), -3 (GPC3), and -5 (GPC5) in oral squamous cell carcinoma (OSCC) and tumor-free lateral margins (TM) and their association with the HH pathway. Quantitative PCR was performed for GPC1, GPC3, GPC5, SHH, PTCH1, SMO, and GLI1 genes in samples of OSCC (n=31), TM (n=12), and non-neoplastic oral mucosa (NNM) of healthy patients (n=6), alongside an immunohistochemical evaluation of GPC1, GPC3, and GPC5 proteins and HH proteins SHH and glioma-associated oncogene homolog 1 (GLI1). Double staining for GPC3/SHH, GPC5/SHH, GPC3/tubulin [ac Lys40], GPC5/Tubulin [ac Lys40], and GPC5/GLI1 was also performed. Overexpression of GPC1 and GPC5 in tumor samples and underexpressed levels of GPC3 gene transcripts were observed when compared with TM (standard sample). HH pathway mRNA aberrant expression in OSCC samples and a negative correlation between GPC1 and GPC5 at transcription levels were detected. GPC1 staining was rare in OSCC, but positive cells were found in NNM and TM. Otherwise positive immunostaining for GPC3 and GPC5 was observed in OSCCs, but not in NNM and TM. Blood vessels adjacent to tumor islands were positive for GPC1 and GPC5. Co-localization of GPC3-positive and GPC5-positive cells with SHH and Tubulin [ac Lys40] proteins was noted, as well as of GPC5 and GLI1. The absence of the GPC1 protein in neoplastic cells, underexpression of the GPC3 gene, and co-localization of GPCs and HH proteins may indicate the maintenance of aberrant HH pathway activation in OSCC.

Abstract A08: Application of Miltuximab®-IRDye800 for near-infrared fluorescence imaging of urothelial carcinoma in vivo

Abstract Introduction and Objectives: Up to 50% of patients with non-muscle invasive bladder cancer suffer from recurrence or progression into a muscle-invasive disease despite multiple resections and long-term chemo and immunotherapy. Possible reasons for that are incomplete resection and reimplantation of cancer cells, which could be prevented by early detection and improved resection of tumor margins. Fluorescence-guided surgery is beneficial in some patients with flat tumors; however, it is currently based on metabolic differences of cancer cells and lacks specificity in many cases. Molecular imaging could improve the specificity of fluorescence-guided surgery since it can target biomarkers expressed by individual tumors. We aimed to select a biomarker and develop a probe for molecular imaging of urothelial carcinoma (UC). Methods: One of the biomarkers present in UC is a heparan sulphate proteoglycan Glypican-1 (GPC-1). The anti-GPC-1 monoclonal antibody Miltuximab (Glytherix Ltd. Sydney, Australia) was used to analyze the expression of GPC-1 in UC cell lines T24 and UM-UC-6. Miltuximab was then labelled by a fluorescent dye IRDye800CW and tested in vivo. Luciferase-transfected UM-UC-6 cells were injected subcutaneously into the hind leg of ten nude mice. When the tumors reached 1000 mm3 in volume, the mice were intravenously injected with 5.7 mg/kg Miltuximab-IRDye800 or isotype IgG-IRDye800. They were then imaged daily for ten days and sacrificed for ex vivo imaging to study the distribution of Miltuximab within the body and its accumulation in the tumors. Results: Flow cytometry and Western blotting demonstrated high expression of GPC-1 in cells UM-UC-6 and T24. UM-UC-6 cells formed subcutaneous tumors that were histologically representative of tumors of human bladder in all animals. From one to ten days after the intravenous injection of the fluorescently labeled Miltuximab, we detected its high accumulation in the tumors by in vivo and ex vivo imaging. One day after the injection, tumors could be visualized in both groups with tumor-to-noise (T/N) ratio of 2. On the 5th day, this value went up to 6.7 for Miltuximab and 3.5 for the Isotype IgG group. By day 10, non-bound antibody molecules further cleared from the mice, resulting in T/N ratio of 14.1 in the mice injected with Miltuximab, while in the control group the value reached only 4.8. Conclusions: Overexpression of GPC-1 in UC shows its high potential as a target for molecular imaging. The animal model developed in this study demonstrated accumulation of the antibody in the tumor and its strong visualization, which could be helpful in tumor detection and resection. Another advantage of this platform is its high potential for clinical translation. IRDye800 can be visualized by existing equipment for indocyanine green imaging and has been proven to be safe in several clinical trials. The antibody Miltuximab has also been used in clinic and has an excellent safety profile. Citation Format: Dmitry Polikarpov, Douglas Campbell, Lucinda McRobb, Alexander Zaslavsky, Andrei Zvyagin, Bradley Walsh, Ganesh Palapattu, David Gillatt. Application of Miltuximab®-IRDye800 for near-infrared fluorescence imaging of urothelial carcinoma in vivo [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A08.

Glypican-1 Promotes Tumorigenesis by Regulating the PTEN/Akt/β-Catenin Signaling Pathway in Esophageal Squamous Cell Carcinoma.

Glypican-1 (GPC1), a cell-surface heparan sulfate proteoglycan, promotes the pathogenesis of many human cancers. This study focuses on the role of GPC1 in the promotion of cell proliferation and motility in esophageal squamous cell carcinoma (ESCC). The expression and distribution of GPC1 were measured in tumor tissues from 248 ESCC patients using immunohistochemical (IHC) assays. Cell counting (kit-8), flow cytometry, Transwell, wound healing, IHC, and Western blotting assays were performed to examine the molecular mechanisms that underlie how GPC1 enhances cell proliferation and motility. The level of GPC1 was higher in ESCC tumor samples than in para-tumor tissues (IHC score: 5.42 ± 2.15 vs. 0.86 ± 0.96). Ectopic overexpression of GPC1 in EC9706 cells promoted cell growth and the G1/S phase transition; conversely, GPC1 knockdown in Eca109 cells attenuated cell proliferation and induced G2/M phase arrest. In addition, GPC1 upregulation enhanced ESCC cell motility and induced epithelial mesenchymal transition (EMT), as demonstrated by the aberrant expression of EMT markers. Mechanistically, we demonstrated that GPC1 increased levels of p-Akt and β-catenin and reduced PTEN expression in ESCC. Our study indicated that GPC1 promotes the aggressive proliferation of ESCC cells by regulating the PTEN/Akt/β-catenin pathway. GPC1 may be a promising target for ESCC treatment.

EVI1 modulates oncogenic role of GPC1 in pancreatic carcinogenesis.

Glypican-1 (GPC1) protein in exosomes was recently identified as a biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analyses and in vitro assays were conducted to assess the usefulness of GPC1 as a PDAC biomarker, to reveal the biological role of GPC1 in pancreatic carcinogenesis, and to ascertain the regulation mechanism of GPC1. An aberrant overexpression of GPC1 protein which is usually absent in normal pancreatic duct, was a widespread marker across the full spectrum of human PDAC precursors, PDAC, and pancreatic cancerous stroma. In intraductal papillary-mucinous neoplasms (IPMNs), GPC1 tended to be positive in gastric-type IPMN. KRAS mutations were found in all GPC1-positive IPMN cases and in one-third of GPC1-negative IPMN cases. In pancreatic cell lines, GPC1 depletion caused remarkable inhibition of cell growth and migration, suggesting its oncogenic roles. GPC1 depletion upregulated the molecules associated with cell cycle arrest in pancreatic cell lines. Furthermore, KRAS and ecotropic viral integration site 1 (EVI1) oncoprotein upregulated GPC1 expression. In a clinical cohort, GPC1 overexpression was not correlated with pancreatic cancer prognosis. Taken together, these findings suggest the necessity of establishing a threshold of GPC1 value for detecting pancreatic malignancy because GPC1 is overexpressed even in low-grade PDAC precursors which do not always become malignant. Our study also reveals a new aspect of pancreatic carcinogenesis: KRAS and EVI1, two important molecules in early phases of pancreatic carcinogenesis, positively regulate GPC1 expression and likely promote pancreatic carcinogenesis.

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

Abstract 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

Altering Glypican-1 levels modulates canonical Wnt signaling during trigeminal placode development

Glypicans are conserved cell surface heparan sulfate proteoglycans expressed in a spatiotemporally regulated manner in many developing tissues including the nervous system. Here, we show that Glypican-1 (GPC1) is expressed by trigeminal placode cells as they ingress and contribute to trigeminal sensory neurons in the chick embryo. Either expression of full-length or truncated GPC1 in vivo causes defects in trigeminal gangliogenesis in a manner that requires heparan sulfate side chains. This leads to either abnormal placodal differentiation or organization, respectively, with near complete loss of the ophthalmic (OpV) trigeminal ganglion in the most severe cases after overexpression of full-length GPC1. Interestingly, modulating GPC1 alters levels of endogenous Wnt signaling activity in the forming trigeminal ganglion, as indicated by Wnt reporter expression. Accordingly, GPC1 overexpression phenocopies Wnt inhibition in causing loss of OpV placodal neurons. Furthermore, increased Wnt activity rescues the effects of GPC1 overexpression. Taken together, these results suggest that appropriate levels of GPC1 are essential for proper regulation of canonical Wnt signaling during differentiation and organization of trigeminal placodal cells into ganglia.

Expression profile analysis of aorta-gonad-mesonephros region-derived stromal cells reveals genes that regulate hematopoiesis

The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis.

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.

Effects of glypican‐1 on turkey skeletal muscle cell proliferation, differentiation and fibroblast growth factor 2 responsiveness

The heparan sulfate proteoglycan, glypican-1, is a low affinity receptor for fibroblast growth factor 2 (FGF2). Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and an inhibitor of differentiation. Heparan sulfate proteoglycans like glypican-1 are required for FGF2 to transduce an intracellular signal. Understanding the role of glypican-1 in the regulation of FGF2-mediated signaling is important in furthering the understanding of the biological processes involved in muscle development and growth. In the current study, a turkey glypican-1 expression vector construct was transfected into turkey myogenic satellite cells resulting in the overexpression of glypican-1. The proliferation, differentiation, and responsiveness to FGF2 were measured in control and transfected cell cultures. The overexpression of glypican-1 in turkey myogenic satellite cells increased both satellite cell proliferation and FGF2 responsiveness, but decreased the rate of differentiation. The current data support glypican-1 modulation of both proliferation and differentiation through an FGF2-mediated pathway.

Glypican-1 as an Abeta binding HSPG in the human brain: its localization in DIG domains and possible roles in the pathogenesis of Alzheimer's disease.

Previous studies have suggested that heparan sulfate proteoglycans (HSPGs) play a role in deposition of beta-amyloid protein (Abeta) in the Alzheimer's disease (AD) brain. In the present study, we demonstrated that glypican-1 can bind fibrillar Abeta, and the binding is mainly mediated by heparan sulfate (HS) chains. Further analysis revealed that glypican-1 is the major HSPG localized in detergent-insoluble glycosphingolipid-enriched (DIG) domains where all machineries for Abeta production exist and Abeta is accumulated as monomeric and oligomeric forms. Immunohistochemical studies demonstrated that glypican-1 is localized in primitive plaques as well as classic plaques. Moreover, overexpression of glypican-1 and amyloid precursor protein in SH-SY5Y cells resulted in reduced cell viability and made cells more susceptible to thapsigargin-induced stress and Abeta toxicity. The results raise the possibility that glypican-1 interacts with oligomerized or polymerized Abeta in such a specific compartment as DIG, resulting not only in amyloid deposition in senile plaques of AD brain, but also in accelerating neuronal cell death in response to stress and Abeta.

Heparan Sulfate Proteoglycans as Regulators of Fibroblast Growth Factor-2 Signaling in Brain Endothelial Cells: SPECIFIC ROLE FOR GLYPICAN-1 IN GLIOMA ANGIOGENESIS

Fibroblast growth factor-2 (FGF2) is a potent angiogenic factor in gliomas. Heparan sulfate promotes ligand binding to receptor tyrosine kinase and regulates signaling. The goal of this study was to examine the contribution of heparan sulfate proteoglycans (HSPGs) to glioma angiogenesis. Here we show that all brain endothelial cell HSPGs carry heparan sulfate chains similarly capable of forming a ternary complex with FGF2 and fibroblast growth factor receptor-1c and of promoting a mitogenic signal. Immunohistochemical analysis revealed that glypican-1 was overexpressed in glioma vessel endothelial cells, whereas this cell-surface HSPG was consistently undetectable in normal brain vessels. To determine the effect of increased glypican-1 expression on FGF2 signaling, we transfected normal brain endothelial cells, which express low base-line levels of glypican-1, with this proteoglycan. Glypican-1 expression enhanced growth of brain endothelial cells and sensitized them to FGF2-induced mitogenesis despite the fact that glypican-1 remained a minor proteoglycan. In contrast, overexpression of syndecan-1 had no effect on growth or FGF2 sensitivity. We conclude that the glypican-1 core protein has a specific role in FGF2 signaling. Glypican-1 overexpression may contribute to angiogenesis and the radiation resistance characteristic of this malignancy.