[Preparation of bacterial outer membrane vesicles with anti-GPC3 single-chain antibody and identification of their targeting effects on hepatocellular carcinoma].

This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo. The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD600=0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 ℃. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs (P=0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.

Pan-cancer analysis and the oncogenic role of Glypican 1 in hepatocellular carcinoma.

Recent studies indicate that Glypican 1 (GPC-1) is aberrantly expressed and plays a key role in certain cancers, but little is known in the hepatocellular carcinoma. Raw data from TCGA, GTEx and TIMER databases were utilized to comprehensively analyze GPC-1 expression landscape in pan-cancer, and the biological function of GPC-1 was investigated in liver cancer cells. The results revealed that GPC-1 is highly expressed in HCC, negatively correlated with survival, and also positively correlated with immune infiltration and clinical stage. Furthermore, GPC-1 promoted cell proliferation and inhibited apoptosis in the HCC cell lines. WGCNA analysis and HCCDB database revealed that Akt acted as a key molecule related to GPC-1, influencing biological functions and regulating cell malignant behaviors via the AKT signaling pathway. In conclusion, our findings provide a relatively comprehensive understanding of the oncogenic role of GPC-1 in HCC, implying that GPC-1 could serve as an innovative therapeutic target.

GPC2 promotes prostate cancer progression via MDK-mediated activation of PI3K/AKT signaling pathway.

Prostate cancer is a major medical problem for men worldwide. Advanced prostate cancer is currently incurable. Recently, much attention was paid to the role of GPC2 in the field of oncology. Nevertheless, there have been no investigations of GPC2 and its regulatory mechanism in prostate cancer. Here, we revealed a novel action of GPC2 and a tumor promoting mechanism in prostate cancer. GPC2 was upregulated in prostate cancer tissues and cell lines. Higher expression of GPC2 was correlated with higher Gleason score, lymphatic metastasis, and worse overall survival in prostate cancer patients. Decreased expression of GPC2 inhibited cell proliferation, migration, and invasion in prostate cancer, whereas GPC2 overexpression promoted these properties. Mechanistically, GPC2 promoted the activation of PI3K/AKT signaling pathway through MDK. The rescue assay results in prostate cancer cells demonstrated that overexpression of MDK could attenuate GPC2 knockdown induced inactivation of PI3K/AKT signaling and partly reverse GPC2 knockdown induced inhibition of cell proliferation, migration, and invasion. In all, our study identified GPC2 as an oncogene in prostate cancer. GPC2 promoted prostate cancer cell proliferation, migration, and invasion via MDK-mediated activation of PI3K/AKT signaling pathway. GPC2 might be a promising prognosis predictor and potential therapeutic target in prostate cancer.

Internalization of PEI-based complexes in transient transfection of HEK293 cells is triggered by coalescence of membrane heparan sulfate proteoglycans like Glypican-4.

Polymer-cationic mediated gene delivery is a well-stablished strategy of transient gene expression (TGE) in mammalian cell cultures. Nonetheless, its industrial implementation is hindered by the phenomenon known as cell density effect (CDE) that limits the cell density at which cultures can be efficiently transfected. The rise in personalized medicine and multiple cell and gene therapy approaches based on TGE, make more relevant to understand how to circumvent the CDE. A rational study upon DNA/PEI complex formation, stability and delivery during transfection of HEK293 cell cultures has been conducted, providing insights on the mechanisms for polyplexes uptake at low cell density and disruption at high cell density. DNA/PEI polyplexes were physiochemically characterized by coupling X-ray spectroscopy, confocal microscopy, cryo-transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR). Our results showed that the ionic strength of polyplexes significantly increased upon their addition to exhausted media. This was reverted by depleting extracellular vesicles (EVs) from the media. The increase in ionic strength led to polyplex aggregation and prevented efficient cell transfection which could be counterbalanced by implementing a simple media replacement (MR) step before transfection. Inhibiting and labeling specific cell-surface proteoglycans (PGs) species revealed different roles of PGs in polyplexes uptake. Importantly, the polyplexes uptake process seemed to be triggered by a coalescence phenomenon of HSPG like glypican-4 around polyplex entry points. Ultimately, this study provides new insights into PEI-based cell transfection methodologies, enabling to enhance transient transfection and mitigate the cell density effect (CDE).

Dysregulation of Glypicans and Notum in Osteoarthritis: Plasma Levels, Bone Marrow Mesenchymal Stromal Cells and Osteoblasts.

In this study of the alterations of Glypicans 1 to 6 (GPCs) and Notum in plasma, bone marrow mesenchymal stromal cells (BM-MSCs) and osteoblasts in Osteoarthritis (OA), the levels of GPCs and Notum in the plasma of 25 patients and 24 healthy subjects were measured. In addition, BM-MSCs from eight OA patients and eight healthy donors were cultured over a period of 21 days using both a culture medium and an osteogenic medium. Protein and gene expression levels of GPCs and Notum were determined using ELISA and qPCR at 0, 7, 14 and 21 days. GPC5 and Notum levels decreased in the plasma of OA patients, while the BM-MSCs of OA patients showed downexpression of GPC6 and upregulation of Notum. A decrease in GPC5 and Notum proteins and an increase in GPC3 were found. During osteogenic differentiation, elevated GPCs 2, 4, 5, 6 and Notum mRNA levels and decreased GPC3 were observed in patients with OA. Furthermore, the protein levels of GPC2, GPC5 and Notum decreased, while the levels of GPC3 increased. Glypicans and Notum were altered in BM-MSCs and during osteogenic differentiation from patients with OA. The alterations found point to GPC5 and Notum as new candidate biomarkers of OA pathology.

Combined genetic-pharmacologic inactivation of tightly linked ADAMTS proteases in temporally specific windows uncovers distinct roles for versican proteolysis and glypican-6 in cardiac development.

Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. We show that matrix-degrading metalloproteases ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each gene have mild cardiac anomalies, however, their combined genetic inactivation to elicit cooperative roles is precluded by tight gene linkage. Therefore, we coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and investigated their potential substrates in mouse cardiac development. ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows spanning myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant versican mutants. Mass spectrometry-based N-terminomics was used to seek relevant substrates. Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which was also observed in mice with ADAMTS cleavage-resistant versican. Combined inactivation after 12.5 days impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect. N-terminomics of combined ADAMTS knockout and control hearts identified a cleaved glypican-6 peptide only in the controls. ADAMTS1 and ADAMTS5 expression in cells was associated with specific glypican-6 cleavages. Paradoxically, combined ADAMTS1 and ADAMTS5 inactivation reduced cardiac glypican-6 and outflow tract Gpc6 transcription. Notably, Gpc6-/- hearts demonstrated similar rotational defects as combined ADAMTS inactivated hearts and both had reduced hedgehog signaling. Thus, versican proteolysis in cardiac jelly at the canonical Glu441-Ala442 site is cooperatively mediated by ADAMTS1 and ADAMTS5 and required for proper ventricular cardiomyogenesis, whereas, reduced glypican-6 after combined ADAMTS inactivation impairs hedgehog signaling, leading to outflow tract malrotation.

Dichotomous Effects of Glypican-4 on Cancer Progression and Its Crosstalk with Oncogenes.

Glypicans are linked to various aspects of neoplastic behavior, and their therapeutic value has been proposed in different cancers. Here, we have systematically assessed the impact of GPC4 on cancer progression through functional genomics and transcriptomic analyses across a broad range of cancers. Survival analysis using TCGA cancer patient data reveals divergent effects of GPC4 expression across various cancer types, revealing elevated GPC4 expression levels to be associated with both poor and favorable prognoses in a cancer-dependent manner. Detailed investigation of the role of GPC4 in glioblastoma and non-small cell lung adenocarcinoma by genetic perturbation studies displays opposing effects on these cancers, where the knockout of GPC4 with CRISPR/Cas9 attenuated proliferation of glioblastoma and augmented proliferation of lung adenocarcinoma cells and the overexpression of GPC4 exhibited a significant and opposite effect. Further, the overexpression of GPC4 in GPC4-knocked-down glioblastoma cells restored the proliferation, indicating its mitogenic effect in this cancer type. Additionally, a survival analysis of TCGA patient data substantiated these findings, revealing an association between elevated levels of GPC4 and a poor prognosis in glioblastoma, while indicating a favorable outcome in lung carcinoma patients. Finally, through transcriptomic analysis, we attempted to assign mechanisms of action to GPC4, as we find it implicated in cell cycle control and survival core pathways. The analysis revealed upregulation of oncogenes, including FGF5, TGF-ß superfamily members, and ITGA-5 in glioblastoma, which were downregulated in lung adenocarcinoma patients. Our findings illuminate the pleiotropic effect of GPC4 in cancer, underscoring its potential as a putative prognostic biomarker and indicating its therapeutic implications in a cancer type dependent manner.

20(S)-Ginsenoside Rh2 inhibits hepatocellular carcinoma by suppressing angiogenesis and the GPC3-mediated Wnt/ß­catenin signaling pathway.

20(S)-Ginsenoside Rh2 has significant anti-tumor effects in various types of cancers, including human hepatocellular carcinoma (HCC). However, its molecular targets and mechanisms of action remain largely unknown. Here, we aim to elucidate the potential mechanisms by which Rh2 suppresses HCC growth. We first demonstrate the role of Rh2 in inhibiting angiogenesis. We observe that Rh2 effectively suppresses cell proliferation and induces apoptosis in HUVECs. Furthermore, Rh2 significantly inhibits HepG2-stimulated HUVEC proliferation, migration and tube formation, accompanied by the downregulation of VEGF and MMP-2 expressions. We also reveal that Rh2 inhibits HCC growth through the downregulation of glypican-3-mediated activation of the Wnt/ß-catenin pathway. We observe a dose-dependent inhibition of proliferation and induction of apoptosis in HepG2 cells upon Rh2 treatment, which is mediated by the inhibition of glypican-3/Wnt/ß-catenin signaling. Moreover, downregulation of glypican-3 expression enhances the effects of Rh2 on the glypican-3/Wnt/ß-catenin signaling pathway, resulting in greater suppression of tumor growth in HepG2 cells. Collectively, our findings shed light on the molecular mechanisms through which Rh2 modulates HCC growth, which involve the regulation of angiogenesis and the glypican-3/Wnt/ß-catenin pathway. These insights may pave the way for the development of novel therapeutic strategies targeting these pathways for the treatment of HCC.

Construction of GPC3-modified Lipopolymer SiRNA Delivery System.

BACKGROUND: Gene therapy has been widely concerned because of its unique therapeutic mechanism. However, due to the lack of safe and effective carries, it has not been widely used in clinical practice. Glypican 3 (GPC3) is a highly specific proteoglycan for hepatocellular carcinoma and is a potential diagnostic and therapeutic target for hepatocellular carcinoma. Herein, to monitor the effect of gene therapy and enhance the transfection efficiency of gene carriers, GPC3-modified lipid polyethyleneimine-modified superparamagnetic nanoparticle (GLPS), a type of visualized carrier for siRNA (small-interfering RNA) targeting the liver, was prepared. METHODS: We performed in vitro gene silencing, cytotoxicity, and agarose gel electrophoresis to identify the optimal GLPS formulation. In vitro MRI and Prussian blue staining verified the liver-targeting function of GLPS. We also analyzed the biocompatibility of GLPS by co-culturing with rabbit red blood cells. Morphological changes were evaluated using HE staining. RESULTS: The GLPS optimal formulation consisted of LPS and siRNA at a mass ratio of 25:1 and LPS and DSPE-PEG-GPC3 at a molar ratio of 2:3. GLPS exhibited evident liver-targeting function. In vitro, we did not observe morphological changes in red blood cells or hemolysis after co-culture. In vivo, routine blood analysis revealed no abnormalities after GLPS injection. Moreover, the tissue morphology of the kidney, spleen, and liver was normal without injury or inflammation. CONCLUSION: GLPS could potentially serve as an effective carrier for liver-targeted MRI monitoring and siRNA delivery.

Simpson-Golabi-Behmel syndrome type 1 with normal birth parameters.

A newborn baby born at 34 weeks and 5 days gestation was admitted for prematurity, dysmorphic features and congenital heart defects. Antenatal scan at 21 weeks showed a large-for-gestational-age foetus with a large abdominal circumference and liver, ventricular septal defect, right prominent renal pelvis and echogenic bowel. Antenatal genetic tests for overgrowth syndromes were negative. The mother had early onset pre-eclampsia. After birth, an overgrowth syndrome was still suspected despite the baby having normal birth parameters. Raw data of the trio whole exome sequencing from the amniocentesis sample were manually inspected. Hemizygous exon 7 deletion in the GPC3 gene was found, and a postnatal diagnosis of Simpson-Golabi-Behmel syndrome, a rare overgrowth syndrome, was made. This case report discusses the significance of antenatal findings, an atypical presentation of a rare syndrome and the obstacles of diagnostic genetic testing.

Extraction-free, immuno-RPA-CRISPR/Cas13a-based one-pot detection of glypican-3 directly from extracellular vesicles.

BACKGROUND: Glypican-3 (GPC3) is a heparan sulfate proteoglycan (HSPG) that binds to the cell membrane via glycosylphosphatidylinositol (GPI). It is not found in healthy adult liver but is overexpressed in human hepatocellular carcinoma (HCC). The protein marker GPC3 on extracellular vesicles (GPC3+ EVs) is also useful for HCC detection. Nevertheless, the absence of practical and dependable quantitative techniques to evaluate EVs proteins prevents their clinical implementation. RESULTS: Here, using an immuno-recombinase polymerase amplification (immuno-RPA) process and dual amplification of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, we firstly create an extraction-free one-pot immuno-RPA-CRISPR (opiCRISPR) for the direct and extremely sensitive detection of EVs proteins. The EVs protein-targeted detection probe is amplified by RPA to generate a long repetitive sequence containing multiple CRISPR RNA (crRNA) targeting barcodes, and the signal is further amplified by the CRISPR-Cas13a side-chain cleavage activity to generate a fluorescent signal. The results show that circulating extracellular vesicle GPC3 (eGPC3) levels are a reliable marker for GPC3 expression in tumor, opening up new avenues for tumor diagnosis. SIGNIFICANCE AND NOVELTY: We created an eGPC3 assay based on the CRISPR-Cas13a system, and successfully study the significance of extracellular vesicle GPC3 markers in hepatocellular carcinoma.

GPC2 as a diagnostic and prognostic marker regulated progression of colorectal cancer.

BACKGROUND AND STUDY AIMS: Glypican 2 (GPC2) is a member of the glypican gene family and is expressed in multiple kinds of cancer. However, the function and mechanism of GPC2 in colorectal cancer remains unclear. In this study, we aimed to identify the role of GPC2 on tumor cell proliferation and survival in colorectal cancer. PATIENTS AND METHODS: Ten pairs of colon cancer and matched normal colon tissues were collected in this research. GEPIA was used to analysis the GPC2 gene expression profile in TGCA data base. RT-qPCR and western blot assay were performed to determine the mRNA and protein expressions. CCK-8, Flow cytometry and colon formation assay were applied to evaluate cell viability. IHC staining was performed to evaluate the protein expression in tissues. The function of GPC2 in vivo was verified by an animal model of colon cancer. RESULTS: Through the bioinformatics analysis and qRT-PCR validation, we found that GPC2 was upregulated in the colon cancer tissues and cells. GPC2 knockdown suppressed cell proliferation in vitro and in vivo was confirmed by the results of CCK-8, colony formation assays, and tumor xenograft models. Moreover, by the analysis of flow cytometry assay and gain-or-loss function experiments, we discovered that CEP164 was highly associated with the expression state of GPC2, and mediated G2/M-phase arrest in GPC2-downregulated tumor cells. CONCLUSION: GPC2 might be a novel oncogenic gene in colorectal cancer, suggesting that it could be a considerable marker for the diagnosis and prognosis of colorectal cancer.

Extracellular Vesicular Analysis of Glypican 1 mRNA and Protein for Pancreatic Cancer Diagnosis and Prognosis.

Detecting pancreatic duct adenocarcinoma (PDAC) in its early stages and predicting late-stage patient prognosis undergoing chemotherapy is challenging. This work shows that the activation of specific oncogenes leads to elevated expression of mRNAs and their corresponding proteins in extracellular vesicles (EVs) circulating in blood. Utilizing an immune lipoplex nanoparticle (ILN) biochip assay, these findings demonstrate that glypican 1 (GPC1) mRNA expression in the exosomes-rich (Exo) EV subpopulation and GPC1 membrane protein (mProtein) expression in the microvesicles-rich (MV) EV subpopulation, particularly the tumor associated microvesicles (tMV), served as a viable biomarker for PDAC. A combined analysis effectively discriminated early-stage PDAC patients from benign pancreatic diseases and healthy donors in sizable clinical from multiple hospitals. Furthermore, among late-stage PDAC patients undergoing chemotherapy, lower GPC1 tMV-mProtein and Exo-mRNA expression before treatment correlated significantly with prolonged overall survival. These findings underscore the potential of vesicular GPC1 expression for early PDAC screenings and chemotherapy prognosis.

Whole-genome sequencing and RNA sequencing analysis reveals novel risk genes and differential expression patterns in hepatoblastoma.

Hepatoblastoma (HB) is an uncommon malignant liver cancer primarily affecting infants and children, characterized by the presence of tissue that resembling fetal hepatocytes, mature liver cells or bile duct cells. The primary symptom in affected children is abdominal lumps. HB constitutes approximately 28% of all liver tumors and two-thirds of liver malignancies in the pediatric and adolescent population. Despite its high prevalence, the underlying mechanism of HB pathogenesis remain largely unknown. To reveal the genetic alternations associated with HB, we conducted a comprehensive genomic study using whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) techniques on five HB patients. We aimed to use WGS to identify somatic variant loci associated with HB, including single nucleotide polymorphisms (SNPs), insertions and deletions (Indels), and copy number variations (CNVs). Notably, we found deleterious mutation in CTNNB1, AXIN2 and PARP1, previously implicated in HB. In addition, we discovered multiple novel genes potentially associated with HB, including BRCA2 and GPC3 which require further functional validation to reveal their contributions to HB development. Furthermore, the American College of Medical Genetics and Genomics (ACMG) analysis identified the ABCC2 gene was the pathogenic gene as a potential risk gene linked with HB. To study the gene expression patterns in HB, we performed RNA-seq analysis and qPCR validation to reveal differential expression of four candidate genes (IGF1R, METTL1, AXIN2 and TP53) in tumors compared to nonneoplastic liver tissue in HB patients (P-Val < 0.01). These findings shed lights on the molecular mechanisms underlying HB development and facilitate to advance future personalized diagnosis and therapeutic interventions of HB.

Differential heparan sulfate dependency of the Drosophila glypicans.

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.

The role and mechanism of HIF-1α-mediated glypican-3 secretion in hypoxia-induced tumor progression in hepatocellular carcinoma.

OBJECTIVE: To explore the expression and secretion mechanism of glypican-3 (GPC3) in hepatocellular carcinoma (HCC) cells under hypoxic conditions, and its role in tumor progression. METHODS: Huh7 cells with and without the knockdown of hypoxia-inducible factor 1-alpha (HIF-1α) were cultured under 1% O2 for varying durations to induce hypoxia. The expression levels of GPC3, HSP70, CD63, STX11 and SYT7 in the cytoplasm and exosomes of Huh7 cells were evaluated by western blotting and immunofluorescence. GPC3 protein expression was further measured in cells treated with GW4869 under hypoxic conditions. Huh7 cells and human umbilical vein endothelial cells (HUVECs) were cultured with the exosomes extracted from the control and GPC3-knockdown cells, the cell proliferation, migration, epithelial-mesenchymal transition (EMT), invasion, and in vitro angiogenesis were analyzed. Tumor xenografts were established to assess the role of GPC3-deficient exosomes in tumor growth. RESULTS: Hypoxic culture conditions downregulated GPC3, STX11 and SYT7 protein levels in the Huh7 cells and upregulated GPC3 mRNA, and also increased GPC3 protein expression in the exosomes. HIF-1α knockdown, as well as treatment with GW4869, upregulated GPC3 protein in the Huh7 cells grown under 1% O2, but downregulated exosomal GPC3. Furthermore, exosomes derived from the GPC3-knockdown cells inhibited the proliferation and migration of Huh7 cells, decreased the expression of N-cadherin, vimentin, ß-catenin, c-Myc and cyclin D1, and increased that of E-cadherin. Likewise, the GPC3-deficient exosomes also suppressed the invasion and tube formation ability of the HUVECs compared to that of control cells. Consistent with the in vitro results, the GPC3-deficient exosomes also repressed tumor growth in vivo. CONCLUSION: Hypoxia promoted secretion of exosomal GPC3 through the activation of HIF-1α. GPC3-deficient exosomes inhibited the proliferation, migration and EMT of HCC cells via the Wnt/ß-catenin signaling pathway, and suppressed the angiogenic potential of HUVECs. This provided a novel understanding of the role of exosomal GPC3 in HCC progression.

Analysis of GATA3 and FOXA2 expression suggests that downregulation of genes involved in the maintenance of a mature yolk sac tumor phenotype may underlie sarcomatoid transformation.

In the post-chemotherapy setting, germ cell tumors of the testis (GCTT) that resemble non-specific sarcomas and co-express cytokeratins and glypican-3 (GPC3) are diagnosed as "sarcomatoid yolk sac tumor postpubertal-type (YSTpt)". The diagnosis of sarcomatoid YSTpt is clinically relevant but challenging due to its rarity, non-specific histology, and negative α-fetoprotein (AFP) staining. Recently, FOXA2 has emerged as a key-gene in the reprogramming of GCTT (activating the transcription of several genes, among which GATA3), and immunohistochemical studies showed that GATA3 and FOXA2 have a higher sensitivity for non-sarcomatoid YSTpt than GPC3 and AFP. We found that sarcomatoid YSTpt did not express FOXA2 [0: 14/14 (100%)] and showed focal expression of GATA3 [0: 12/14 (85.7%), 1 + : 2/14 (14.3%)], thus suggesting that these markers are not useful in diagnosing this tumor. Furthermore, we proposed a potential mechanism of sarcomatoid transformation in the post-chemotherapy setting of GCTT, mediated by the downregulation of FOXA2 and GATA3.

Biological and clinical significance of the glypican-3 gene in human lung adenocarcinoma: An in silico analysis.

Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan, has long been found to be dysregulated in human lung adenocarcinomas (LUADs). Nevertheless, the function, mutational profile, epigenetic regulation, co-expression profile, and clinicopathological significance of the GPC3 gene in LUAD progression are not well understood. In this study, we analyzed cancer microarray datasets from publicly available databases using bioinformatics tools to elucidate the above parameters. We observed significant downregulation of GPC3 in LUAD tissues compared to their normal counterparts, and this downregulation was associated with shorter overall survival (OS) and relapse-free survival (RFS). Nevertheless, no significant differences in the methylation pattern of GPC3 were observed between LUAD and normal tissues, although lower promoter methylation was observed in male patients. GPC3 expression was also found to correlate significantly with infiltration of B cells, CD8+, CD4+, macrophages, neutrophils, and dendritic cells in LUAD. In addition, a total of 11 missense mutations were identified in LUAD patients, and ~1.4% to 2.2% of LUAD patients had copy number amplifications in GPC3. Seventeen genes, mainly involved in dopamine receptor-mediated signaling pathways, were frequently co-expressed with GPC3. We also found 11 TFs and 7 miRNAs interacting with GPC3 and contributing to disease progression. Finally, we identified 3 potential inhibitors of GPC3 in human LUAD, namely heparitin, gemcitabine and arbutin. In conclusion, GPC3 may play an important role in the development of LUAD and could serve as a promising biomarker in LUAD.