Unconventional secretion mediated by direct protein self-translocation across the plasma membranes of mammalian cells.

In recent years, a surprisingly complex picture emerged about endoplasmic reticulum (ER)/Golgi-independent secretory pathways, and several routes have been discovered that differ with regard to their molecular mechanisms and machineries. Fibroblast growth factor 2 (FGF2) is secreted by a pathway of unconventional protein secretion (UPS) that is based on direct self-translocation across the plasma membrane. Building on previous research, a component of this process has been identified to be glypican-1 (GPC1), a GPI-anchored heparan sulfate proteoglycan located on cell surfaces. These findings not only shed light on the molecular mechanism underlying this process but also reveal an intimate relationship between FGF2 and GPC1 that might be of critical relevance for the prominent roles they both have in tumor progression and metastasis.

Interplay between APP and glypican-1 processing and α-synuclein aggregation in undifferentiated and differentiated human neural progenitor cells.

In Parkinson's disease, there is an accumulation of α-synuclein (SYN) aggregates in neurons, which is promoted by neuroinflammation. In neural cells, cytokine-induced SYN aggregation is modulated by heparan sulfate (HS) derived from glypican-1 (GPC1) by amyloid precursor protein (APP) and nitric oxide (NO)-dependent cleavage. We have explored possible interplay between APP, GPC1, and SYN in undifferentiated and differentiated neural progenitor cells (NPCs) by modulating APP and GPC1 processing. Effects were monitored by immunofluorescence microscopy and slot immunoblotting using antibodies recognizing APP degradation products, HS released from GPC1, and SYN aggregates (filamentous SYN [SYNfil]). Suppression of HS release from GPC1 by inhibition of ß-secretase or by NO deprivation resulted in no or slight increase in SYNfil aggregation. Stimulation of HS release by ascorbate did not further increase SYNfil staining. Interleukin-6 (IL-6) induced increased APP and GPC1 processing and SYNfil formation, which was reduced when ß-secretase was inhibited and when HS release was impeded by NO deprivation. Ascorbate restored APP and GPC1 processing but did not affect SYNfil formation. Ascorbate-dependent differentiation of NPC resulted in the expression of tyrosine hydroxylase (TH) which colocalized with SYNfil. Suppression of APP processing by inhibition of ß-secretase greatly disturbed the differentiation process. IL-6 induced coclustering of APP-degradation products, TH, HS, and SYNfil, which could be reversed by stimulation of HS release from GPC1 by excess ascorbate. We suggest that continuous release of HS from GPC1 moderates SYN aggregation and supports differentiation of NPC to dopaminergic neurons.

Pharmacokinetics and Biodistribution of 89Zr-Miltuximab and Its Antibody Fragments as Glypican-1 Targeting Immuno-PET Agents in Glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain cancer, accounting for about 85% of all primary central nervous system (CNS) tumors. With standard treatment strategies like surgery, radiation, and chemotherapy, the median survival time of patients with GBM is only 12-15 months from diagnosis. The poor prognosis of GBM is due to a very high tumor recurrence rate following initial treatment, indicating a dire need for improved diagnostic and therapeutic alternatives for this disease. Antibody-based immunotheranostics holds great promise in treating GBM, combining the theranostic applications of radioisotopes and target-specificity of antibodies. In this study, we developed and validated antibody-based positron emission tomography (PET) tracers targeting the heparan sulfate proteoglycan, glypican-1 (GPC-1), for noninvasive detection of disease using diagnostic molecular imaging. GPC-1 is overexpressed in multiple solid tumor types, including GBM, and is a promising biomarker for novel immunotheranostics. Here, we investigate zirconium-89 (89Zr)-conjugated Miltuximab (a clinical stage anti-GPC-1 monoclonal antibody developed by GlyTherix, Ltd.) and engineered fragments for their potential as immuno-PET tracers to detect GPC-1positive GBM tumors in preclinical models. We explore the effects of molecular size, avidity, and Fc-domain on the pharmacokinetics and biodistribution in vivo, by comparing in parallel the full-length antibody (Miltuximab), Fab'2, Fab, and single-chain variable fragment (scFv) formats. High radiolabeling efficiency (>95%) was demonstrated by all the formats and the stability post-radiolabeling was higher for larger constructs of Miltuximab and the Fab. Receptor-mediated internalization of all 89Zr-labeled formats was observed in a human GBM cell line in vitro, while full-length Miltuximab demonstrated the highest tumor retention (5.7 ± 0.94% ID/g, day-9 postinjection (p.i.)) and overall better tumor-to-background ratios than the smaller Fc-less formats. Results from in vivo PET image quantification and ex vivo scintillation counting were highly correlated. Altogether, 89Zr-DFO-Miltuximab appears to be an effective immuno-PET imaging agent for detecting GPC-1positive tumors such as GBM and the current results support utility of the Fc containing whole mAb format over smaller antibody fragments for this target.

The Pulmonary Endothelial Glycocalyx Modifications in Glypican 1 Knockout Mice Do Not Affect Lung Endothelial Function in Physiological Conditions.

The endothelial glycocalyx is a dynamic signaling surface layer that is involved in the maintenance of cellular homeostasis. The glycocalyx has a very diverse composition, with glycoproteins, proteoglycans, and glycosaminoglycans interacting with each other to form a mesh-like structure. Due to its highly interactive nature, little is known about the relative contribution of each glycocalyx constituent to its overall function. Investigating the individual roles of the glycocalyx components to cellular functions and system physiology is challenging, as the genetic manipulation of animals that target specific glycocalyx components may result in the development of a modified glycocalyx. Thus, it is crucial that genetically modified animal models for glycocalyx components are characterized and validated before the development of mechanistic studies. Among the glycocalyx components, glypican 1, which acts through eNOS-dependent mechanisms, has recently emerged as a player in cardiovascular diseases. Whether glypican 1 regulates eNOS in physiological conditions is unclear. Herein, we assessed how the deletion of glypican 1 affects the development of the pulmonary endothelial glycocalyx and the impact on eNOS activity and endothelial function. Male and female 5-9-week-old wild-type and glypican 1 knockout mice were used. Transmission electron microscopy, immunofluorescence, and immunoblotting assessed the glycocalyx structure and composition. eNOS activation and content were assessed by immunoblotting; nitric oxide production was assessed by the Griess reaction. The pulmonary phenotype was evaluated by histological signs of lung injury, in vivo measurement of lung mechanics, and pulmonary ventilation. Glypican 1 knockout mice showed a modified glycocalyx with increased glycocalyx thickness and heparan sulfate content and decreased expression of syndecan 4. These alterations were associated with decreased phosphorylation of eNOS at S1177. The production of nitric oxides was not affected by the deletion of glypican 1, and the endothelial barrier was preserved in glypican 1 knockout mice. Pulmonary compliance was decreased, and pulmonary ventilation was unaltered in glypican 1 knockout mice. Collectively, these data indicate that the deletion of glypican 1 may result in the modification of the glycocalyx without affecting basal lung endothelial function, validating this mouse model as a tool for mechanistic studies that investigate the role of glypican 1 in lung endothelial function.

Complex modulation of cytokine-induced α-synuclein aggregation by glypican-1-derived heparan sulfate in neural cells.

In Parkinson's disease (PD), there is accumulation of α-synuclein (SYN) aggregates in neurons, which is promoted by neuroinflammation. The cytokines TNF-α, IL-1ß and IL-6 induce accumulation of degradation products of the amyloid precursor protein (APP) combined with heparan sulfate (HS) chains released from glypican-1 (Gpc-1) by NO-dependent cleavage. We have investigated the effects of the cytokines and HS on SYN aggregation and secretion in dividing human neuroblastoma (SH-SY5Y) and inducible neural progenitor cells (NPC) by using immunofluorescence microscopy, vesicle isolation and slot blotting with antibodies recognizing SYN monomers and aggregates, Gpc-1, the released HS, endosomes, and autophagosomes. In SH-SY5Y cells, the capacity to release HS was fully utilized, while NPC displayed dormant capacity. TNF-α induced increased formation of SYN aggregates and clustering of HS in SH-SY5Y cells. When the supply of NO was simultaneously increased, SYN and HS accumulation disappeared. When NO formation was inhibited, SYN and HS aggregation also disappeared, but there was now a 4-fold increase in SYN secretion. In NPC, IL-6 induced increased aggregation of SYN and stimulated HS release from Gpc-1. Both SYN and HS co-localized with autophagosome marker. When HS-deficient Gpc-1 was simultaneously generated, by using a cyanobacterial neurotoxin, accumulation diminished and there was massive secretion of SYN. We suggest that the cytokines increase APP processing, which initiates NO-dependent release of HS from Gpc-1. The APP degradation products also trigger SYN aggregation. As HS can inhibit APP processing, HS- or NO-deficiency may result in autophagosomal dysfunction and both APP degradation products and SYN are secreted.

Role of glypican-1 in regulating multiple cellular signaling pathways.

Glypican-1 (GPC1) is one of the six glypican family members in humans. It is composed of a core protein with three heparan sulfate chains and attached to the cell membrane by a glycosyl-phosphatidylinositol anchor. GPC1 modulates various signaling pathways including fibroblast growth factors (FGF), vascular endothelial growth factor-A (VEGF-A), transforming growth factor-ß (TGF-ß), Wnt, Hedgehog (Hh), and bone morphogenic protein (BMP) through specific interactions with pathway ligands and receptors. The impact of these interactions on signaling pathways, activating or inhibitory, is dependent upon specific GPC1 domain interaction with pathway components, as well as cell surface context. In this review, we summarize the current understanding of the structure of GPC1, as well as its role in regulating multiple signaling pathways. We focus on the functions of GPC1 in cancer cells and how new insights into these signaling processes can inform its translational potential as a therapeutic target in cancer.

Reversal of apolipoprotein E4-dependent or chemical-induced accumulation of APP degradation products by vitamin C-induced release of heparan sulfate from glypican-1.

The Apolipoprotein E4 (ApoE4) genotype is the most influential risk factor for sporadic Alzheimer's disease. It appears to be associated with retarded endosome-to-autophagosome trafficking. The amyloid precursor protein (APP) and the heparan sulfate (HS)-containing proteoglycan glypican-1 (Gpc-1) are both processed in endosomes, and mutually regulated by the APP degradation products and the released HS. We have investigated APP and Gpc-1 processing in ApoE3 and ApoE4 expressing human fibroblasts, in human neural stem cells (NSC) exposed to the cholesterol transport inhibitor U18666A and in induced neurons obtained by reprogramming of ApoE fibroblasts (ApoE-iN). We have used immunofluorescence microscopy, flow cytometry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis western blotting with antibodies recognizing the released HS, APP, amyloid ß(Aß), late endosomes (Rab7), autophagosomes (LC3) and neurons (Tuj1). We found that the capacity to release HS was not fully utilized in ApoE4 expressing fibroblasts and that HS-Aß complexes accumulated in the nuclei. In ApoE3 fibroblasts, the ß-cleaved APP C-terminal fragment (ß-CTF) and Aß were primarily present in late endosomes and autophagosomes. When HS release from Gpc-1 was enhanced by ascorbate in ApoE4/4 fibroblasts, there was efficient transfer of Aß and HS from the nuclei to autophagosomes. In U18666A-treated NSC as well as in ApoE4/4-iN we repeatedly found accumulation of APP degradation products (ß-CTF/Aß). This was reversed by subsequent exposure to ascorbate or dehydroascorbic acid.

Glypican 1 and syndecan 1 differently regulate noradrenergic hypertension development: Focus on IP3R and calcium.

BACKGROUND: Vascular dysfunction is a checkpoint to the development of hypertension. Heparan sulfate proteoglycans (HSPG) participate in nitric oxide (NO) and calcium signaling, key regulators of vascular function. The relationship between HSPG-mediated NO and calcium signaling and vascular dysfunction has not been explored. Likewise, the role of HSPG on the control of systemic blood arterial pressure is unknown. Herein, we sought to determine if the HSPG syndecan 1 and glypican 1 control systemic blood pressure and the progression of hypertension. PURPOSE: To determine the mechanisms whereby glypican 1 and syndecan 1 regulate vascular tone and contribute to the development of noradrenergic hypertension. EXPERIMENTAL APPROACH AND KEY RESULTS: By assessing systemic arterial blood pressure we observed that syndecan 1 (Sdc1-/-) and glypican 1 (Gpc1-/-) knockout mice show a similar phenotype of decreased systolic blood pressure that is presented in a striking manner in the Gpc1-/- strain. Gpc1-/- mice are also uniquely protected from a norepinephrine hypertensive challenge failing to become hypertensive. This phenotype was associated with impaired calcium-dependent vasoconstriction and altered expression of calcium-sensitive proteins including SERCA and calmodulin. In addition, Gpc1-/- distinctively showed decreased IP3R activity and increased calcium storage in the endoplasmic reticulum. CONCLUSIONS AND IMPLICATIONS: Glypican 1 is a trigger for the development of noradrenergic hypertension that acts via IP3R- and calcium-dependent signaling pathways. Glypican 1 may be a potential target for the development of new therapies for resistant hypertension or conditions where norepinephrine levels are increased.

Highly sensitive and portable mRNA detection platform for early cancer detection.

Pancreatic cancer, at unresectable advanced stages, presents poor prognoses, which could be prevented by early pancreatic cancer diagnosis methods. Recently, a promising early-stage pancreatic cancer biomarker, extracellular vesicles (EVs) related glypican-1 (GPC1) mRNA, is found to overexpress in pancreatic cancer cells. Current mRNA detection methods usually require expensive machinery, strict preservation environments, and time-consuming processes to guarantee detection sensitivity, specificity, and stability. Herein, we propose a novel two-step amplification method (CHAGE) via the target triggered Catalytic Hairpin Assembly strategy combined with Gold-Enhanced point-of-care-testing (POCT) technology for sensitive visual detection of pancreatic cancer biomarker. First, utilizing the catalyzed hairpin DNA circuit, low expression of the GPC1 mRNA was changed into amplification product 1 (AP1, a DNA duplex) as the next detection targets of the paper strips. Second, the AP1 was loaded onto a lateral flow assay and captured with the gold signal nanoparticles to visualize results. Finally, the detected results can be further enhanced by depositing gold to re-enlarge the sizes of gold nanoparticles in detection zones. As a result, the CHAGE methodology lowers the detection limit of mRNA to 100 fM and provides results within 2 h at 37 °C. Furthermore, we demonstrate the successful application in discriminating pancreatic cancer cells by analyzing EVs' GPC1 mRNA expression levels. Hence, the CHAGE methodology proposed here provides a rapid and convenient POCT platform for sensitive detection of mRNAs through unique probes designs (COVID, HPV, etc.).

Glypican-1 as a target for fluorescence molecular imaging of bladder cancer.

OBJECTIVES: To investigate whether anti-glypican-1 antibody Miltuximab conjugated with near-infrared dye IRDye800CW can be used for in vivo fluorescence imaging of urothelial carcinoma. METHODS: The conjugate, Miltuximab-IRDye800CW, was produced and characterized by size exclusion chromatography and flow cytometry with glypican-1-expressing cells. Balb/c nude mice bearing subcutaneous urothelial carcinoma xenografts were intravenously injected with Miltuximab-IRDye800CW or control IgG-IRDye800CW and imaged daily by fluorescence imaging. After 10 days, tumors and major organs were collected for ex vivo study of the conjugate biodistribution, including its accumulation in the tumor. RESULTS: The intravenous injection of Miltuximab-IRDye800CW to tumor-bearing mice showed its specific accumulation in the tumors with the tumor-to-background ratio of 12.7 ± 2.4, which was significantly higher than that in the control group (4.6 ± 0.9, P < 0.005). The ex vivo imaging was consistent with the in vivo findings, with tumors from the mice injected with Miltuximab-IRDye800CW being significantly brighter than the organs or the control tumors. CONCLUSIONS: The highly specific accumulation and retention of Miltuximab-IRDye800CW in glypican-1-expressing tumors in vivo shows its high potential for fluorescence imaging of urothelial carcinoma and warrants its further investigation toward clinical translation.

Proinflammatory cytokines induce accumulation of glypican-1-derived heparan sulfate and the C-terminal fragment of ß-cleaved APP in autophagosomes of dividing neuronal cells.

Proinflammatory cytokines stimulate expression of ß-secretase, which increases processing of amyloid precursor protein (APP), ultimately leading to the deposition of amyloid beta (Aß). The N-terminal domain of ß-cleaved APP supports Cu/NO-dependent release of heparan sulfate (HS) from the glypican-1 (Gpc-1) proteoglycan. HS is an inhibitor of ß-secretase, thereby constituting a regulatory, negative feedback loop. Here, we have investigated the effect of the proinflammatory cytokines TNF-α, IL-1ß and IL-6 on the interplay between APP processing and release of HS from Gpc-1 in neuronal cells. We have used deconvolution immunofluorescence microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a panel of monoclonal/polyclonal antibodies recognizing the released HS, the N-terminus of Aß, Aß, the C-terminus of APP and the autophagosome marker LC3 as well as the chemical lysosome marker LysoTrackerRed (LTR). We repeatedly found that N2a neuroblastoma cells and human neural stem cells grown in the presence of the cytokines developed large cytoplasmic clusters, which stained positive for HS, the N-terminus of Aß, Aß, the C-terminus of APP, LC3 and LTR, indicating accumulation of HS and APP/APP degradation products in enlarged autophagosomes/lysosomes. The SDS-PAGE of immunoisolates obtained from TNF-α-treated N2a cells by using anti-C-terminus of APP revealed the presence of SDS-stable complexes between HS and the C-terminal fragment of ß-cleaved APP (ßCTF) migrating in the range 10-18 kDa. Clustered accumulation of ßCTF disappeared when HS release was prevented and slightly enhanced when HS release was increased. Hence, when proinflammatory cytokines induce increased processing of APP, inhibition of ß-secretase by HS is insufficient, which may lead to the impaired autophagosomal degradation.

A bispecific T cell engager targeting Glypican-1 redirects T cell cytolytic activity to kill prostate cancer cells.

BACKGROUND: Glypican-1 is a heparan sulfate proteoglycan that is overexpressed in prostate cancer (PCa), and a variety of solid tumors. Importantly, expression is restricted in normal tissue, making it an ideal tumor targeting antigen. Since there is clinical and preclinical evidence of the efficacy of Bispecific T cell Engager (BiTE) therapy in PCa, we sought to produce and test the efficacy of a GPC-1 targeted BiTE construct based on the Miltuximab® sequence. Miltuximab® is a clinical stage anti-GPC-1 antibody that has proven safe in first in human trials. METHODS: The single chain variable fragment (scFv) of Miltuximab® and the CD3 binding sequence of Blinatumomab were combined in a standard BiTE format. Binding of the construct to immobilised recombinant CD3 and GPC-1 antigens was assessed by ELISA and BiaCore, and binding to cell surface-expressed antigens was measured by flow cytometry. The ability of MIL-38-CD3 to activate T cells was assessed using in vitro co-culture assays with tumour cell lines of varying GPC-1 expression by measurement of CD69 and CD25 expression, before cytolytic activity was assessed in a similar co-culture. The release of inflammatory cytokines from T cells was measured by ELISA and expression of PD-1 on the T cell surface was measured by flow cytometry. RESULTS: Binding activity of MIL-38-CD3 to both cell surface-expressed and immobilised recombinant GPC-1 and CD3 was retained. MIL-38-CD3 was able to mediate the activation of peripheral blood T cells from healthy individuals, resulting in the release of inflammatory cytokines TNF and IFN-g. Activation was reliant on GPC-1 expression as MIL-38-CD3 mediated only low level T cell activation in the presence of C3 cells (constitutively low GPC-1 expression). Activated T cells were redirected to lyse PCa cell lines PC3 and DU-145 (GPC-1 moderate or high expression, respectively) but could not kill GPC-1 negative Raji cells. The expression of PD-1 was up-regulated on the surface of MIL-38-CD3 activated T cells, suggesting potential for synergy with checkpoint inhibition. CONCLUSIONS: This study reports preclinical findings into the efficacy of targeting GPC-1 in PCa with BiTE construct MIL-38-CD3. We show the specificity and efficacy of the construct, supporting its further preclinical development.

Exosomal glypican-1 for risk stratification of pancreatic cystic lesions: A case of pathological progression in the absence of any suspicious imaging finding.

The clinical management of patients with pancreatic cystic lesions is of utmost importance to identify those at high risk for pathological progression. Current recommendations are guided by clinical presentation and radiologic criteria, but the results fall short for a disease that the only curative option is surgical resection. There is an urgent need for the introduction of biomarkers that can help in risk assessment of such lesions. We report a case of a pancreatic cystic lesion without imagiological findings suggestive of advanced disease, and high levels of a circulating biomarker, glypican-1 (GPC-1), which parallel those of patients with pancreatic cancer. One year after, the patient revealed malignant progression at follow-up. Our report is unprecedented in the literature. It describes a clinical case in which a biomarker was positive for a patient that only showed progression one year after its detection. This clinical information goes beyond the current knowledge in the field because it shows that the introduction of liquid biopsy and biomarkers is a highly promising clinical tool for the non-invasive assessment of pancreatic cancer precursor lesions, ultimately increasing the rate of patients eligible for surgical resection.

The cyanobacterial neurotoxin ß-N-methylamino-l-alanine prevents addition of heparan sulfate to glypican-1 and increases processing of amyloid precursor protein in dividing neuronal cells.

The neurotoxin ß-N-methylamino-l-alanine replaces l-serine in proteins and produces Alzheimer-like pathology. In proteoglycans, e.g. glypican-1, this should preclude substitution with heparan sulfate chains. Reduced release of heparan sulfate should increase ß-secretase activity and processing of amyloid precursor protein. Cultured cells were treated with ß-N-methylamino-l-alanine during the growth-phase and the effect on heparan sulfate substitution and amyloid precursor protein processing was evaluated using antibodies specific for heparan sulfate, the N- and C-termini of the C-terminal fragment of ß-cleaved amyloid precursor protein, and amyloid beta followed by immunofluorescence microscopy, flow cytometry or SDS-PAGE. Mouse fibroblasts, N2a neuroblastoma cells and human neural stem cells released less heparan sulfate when grown in the presence of ß-N-methylamino-l-alanine. Cells expressing a recombinant, anchor-less glypican-1 secreted heparan sulfate-deficient glypican-1. There was increased processing of amyloid precursor protein in N2a cells when grown in the presence of the neurotoxin. The degradation products accumulated in cytoplasmic clusters. Secretion of amyloid beta increased approx. 3-fold. Human neural stem cells also developed cytoplasmic clusters containing degradation products of amyloid precursor protein. When non-dividing mouse N2a cells or cortical neurons were exposed to ß-N-methylamino-l-alanine there was no effect on heparan sulfate substitution in glypican-1 or on amyloid precursor protein processing.

The Role of Glypican-1 in the Tumour Microenvironment.

Glypican-1 (GPC-1) is a cell surface heparan sulphate proteoglycan that is critical during normal development, but which is not required for normal homoeostasis in the adult. It is, however, overexpressed in a variety of solid tumours and is known to regulate tumour growth, invasion, metastasis and progression, through modulation of tumour cell biology as well as influence on the tumour microenvironment (TME). The role of GPC-1 in the TME and on the tumour cell is broad, as GPC-1 regulates signalling by several growth factors, including FGF, HGF, TGF-ß, Wnt and Hedgehog (Hh). Signalling via these pathways promotes tumour growth and invasive and metastatic ability (drives epithelial-to-mesenchymal transition (EMT)) and influences angiogenesis, affecting both tumour and stromal cells. Broad modulation of the TME via inhibition of GPC-1 may represent a novel therapeutic strategy for inhibition of tumour progression. Here, we discuss the complex role of GPC-1 in tumour cells and the TME, with discussion of potential therapeutic targeting strategies.

Common traffic routes for imported spermine and endosomal glypican-1-derived heparan sulfate in fibroblasts.

Import of the polyamine spermine from the extracellular environment depends on the presence of cell surface heparan sulfate proteoglycans, such as glypican-1. This proteoglycan is internalized by endocytosis, releases its heparan sulfate chains in endosomes by a nitric oxide-, copper- and amyloid precursor protein-dependent mechanism, then penetrates the membrane and is transported to the nucleus and then to autophagosomes. This process is spontaneous or induced by ascorbate depending on the growth-state of the cell. Here, we have explored possible connections between the heparan sulfate traffic route and spermine uptake and delivery in wild-type and Tg2576 mouse fibroblasts. Cells were examined by deconvolution immunofluorescence microscopy. The antibodies used were specific for spermine, glypican-1-derived heparan sulfate, Rab7, nucleolin and a marker for autophagosomes. Endogenous immunostainable spermine was primarily associated with autophagosomes. When spermine synthesis was inhibited, imported spermine appeared in Rab7-positive endosomes. When ascorbate was added, heparan sulfate and spermine were transported to the nucleus where they colocalized with nucleolin. Spermine also appeared in autophagosomes. In a pulse-chase experiment, heparan sulfate and spermine were first arrested in late endosomes by actinomycin D treatment. During the chase, when arrest was abolished, heparan sulfate and spermine were both transported to the nucleus and targeted nucleolin. In amyloid precursor protein-/--fibroblasts, ascorbate failed to induce release of heparan sulfate and spermine remained in the endosomes. We propose that cell surface glypican-1 carries spermine to the endosomes and that the released heparan sulfate carries spermine across the membrane into the cytosol and then to the nucleus.

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

BACKGROUND AND AIMS: 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). METHODS: 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. RESULTS: 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 ß-catenin and reduced PTEN expression in ESCC. CONCLUSIONS: Our study indicated that GPC1 promotes the aggressive proliferation of ESCC cells by regulating the PTEN/Akt/ß-catenin pathway. GPC1 may be a promising target for ESCC treatment.

Anti-glypican-1 antibody-drug conjugate exhibits potent preclinical antitumor activity against glypican-1 positive uterine cervical cancer.

Glypican-1 (GPC1) is highly expressed in solid tumors, especially squamous cell carcinomas (SCCs), and is thought to be associated with disease progression. We explored the use of a GPC1-targeted antibody-drug conjugate (ADC) as a novel treatment for uterine cervical cancer. On immunohistochemical staining, high expression levels of GPC1 were detected in about 50% of uterine cervical cancer tissues and also in a tumor that had relapsed after chemoradiotherapy. Novel anti-GPC1 monoclonal antibodies were developed, and clone 01a033 was selected as the best antibody for targeted delivery of the cytotoxic agent monomethyl auristatin F (MMAF) into GPC1-positive cells. The anti-GPC1 antibody was conjugated with MMAF. On flow cytometry, HeLa and ME180 cervical cancer cells highly expressed GPC1, however, RMG-I ovarian clear cell cancer cell line showed weak expression. The GPC1-ADC was rapidly internalized into GPC1-expressing cells in vitro and was potently cytotoxic to cancer cells highly expressing GPC1. There were no inhibitory effects on cancer cells with low expression of GPC1. In a murine xenograft model, GPC1-ADC also had significant and potent tumor growth inhibition. GPC1-ADC-mediated G2/M phase cell cycle arrest was detected, indicating that the dominant antitumor effect in vivo was MMAF-mediated. The toxicity of GPC-ADC was tolerable within the therapeutic dose range in mice. Our data showed that GPC1-ADC has potential as a promising therapy for uterine cervical cancer.

Heparan sulfate accumulation and perlecan/HSPG2 up-regulation in tumour tissue predict low relapse-free survival for patients with glioblastoma.

Glycosaminoglycans are major components of brain extracellular matrix (ECM), although heparan sulfate (HS) contribution in brain physiology and carcinogenesis remains underinvestigated. This study examined HS content and distribution in glioblastoma multiforme (GBM) tissues in the context of potential molecular mechanisms underlying its deregulation in brain tumours. Totally, 42 tissue samples and paraffin-embedded tissues for 31 patients with different prognosis were investigated. HS expression was demonstrated in 50-55% of the GBM tumours by immunohistochemistry (IHC), while almost no HS content was detected in the surrounding paratumourous brain tissues. Heterogeneous HS distribution in the HS-positive tumours was more related to the necrosis or glandular-like brain zones rather than glioma cells with high or low Ki-67 index. According the Kaplan-Meier curves, HS accumulation in glioma cells was associated with low relapse-free survival (RS) of the GBM patients (p < 0.05) and was likely to be due to the increased transcriptional activity of HSPG core proteins (syndecan-1, 2-3 fold; glypican-1, 2,5 fold; perlecan/HSPG2, 13-14 fold). Activation of perlecan/HSPG2 expression correlated with the patients' survival according Kaplan-Meier (p = 0.0243) and Cox proportional-hazards regression (HR = 3.1; P(Y) = 0.03) analyses, while up-regulation of syndecan-1 and glypican-1 was not associated with the patients survival. Taken together, the results indicate that increase of HS content and up-regulation of perlecan/HSPG2 expression in glioblastoma tissues contribute to tumour development through the transformation of brain extracellular matrix into tumour microenvironment, and represent negative prognostic factors for glioblastoma progression.

Cytochrome b561, copper, ß-cleaved amyloid precursor protein and niemann-pick C1 protein are involved in ascorbate-induced release and membrane penetration of heparan sulfate from endosomal S-nitrosylated glypican-1.

Ascorbate-induced release of heparan sulfate from S-nitrosylated heparan sulfate proteoglycan glypican-1 takes place in endosomes. Heparan sulfate penetrates the membrane and is transported to the nucleus. This process is dependent on copper and on expression and processing of the amyloid precursor protein. It remains unclear how exogenously supplied ascorbate can generate HS-anMan in endosomes and how passage through the membrane is facilitated. Here we have examined wild-type, Alzheimer Tg2576 and amyloid precursor protein (-/-) mouse fibroblasts and human fetal and Niemann-Pick C1 fibroblasts by using deconvolution immunofluorescence microscopy, siRNA technology and [S35]sulfate-labeling, vesicle isolation and gel chromatography. We found that ascorbate-induced release of heparan sulfate was dependent on expression of endosomal cytochrome b561. Formation and nuclear transport of heparan sulfate was suppressed by inhibition of ß-processing of the amyloid precursor protein and formation was restored by copper (I) ions. Membrane penetration was not dependent on amyloid beta channel formation. Inhibition of endosomal exit resulted in accumulation of heparan sulfate in vesicles that exposed the C-terminal of the amyloid precursor protein externally. Endosome-to-nucleus transport was also dependent on expression of the Niemann-Pick C1 protein. We propose that ascorbate is taken up from the medium and is oxidized by cytochrome b561 which, in turn, reduces copper (II) to copper (I) present in the N-terminal, ß-cleaved domain of the amyloid precursor protein. Re-oxidation of copper (I) is coupled to reductive, deaminative release of heparan sulfate from glypican-1. Passage through the membrane may be facilitated by the C-terminal, ß-cleaved fragment of the amyloid precursor protein and the Niemann-Pick C1 protein.