Glypican-3 Protein Expression Research Articles

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

ObjectiveTo explore the expression and secretion mechanism of glypican-3 (GPC3) in hepatocellular carcinoma (HCC) cells under hypoxic conditions, and its role in tumor progression. MethodsHuh7 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. ResultsHypoxic 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, β-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. ConclusionHypoxia 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/β-catenin signaling pathway, and suppressed the angiogenic potential of HUVECs. This provided a novel understanding of the role of exosomal GPC3 in HCC progression.

Expression of Glypican-3 (GPC3) in Malignant and Non-malignant Human Breast Tissues

Specific reports have linked GPC3 with cancer. Its usefulness as a marker has been proved for hepatocarcinoma, melanoma and ovary carcinoma. However, there are no studies analyzing GPC3 usefulness as a biomarker in mammary tumors. The aim of this work was to analyze GPC3 expression in breast tissues and to determine whether it might be useful as a biomarker in breast cancer patients. Expression level of GPC3 mRNA in Brazilian and Argentine human breast tumor (n=121) and peritumoral tissue (n=77) samples was analyzed using qRT-PCR. GPC3 protein expression was analyzed from 69 breast cancer and 10 peritumoral samples using IHC. Statistical analyses were done to evaluate the clinical-pathological significance of GPC3 expression. We found that Brazilian and Argentine populations are statistically different regarding GPC3 mRNA expression. In Argentine patients a lower GPC3 mRNA expression was found in tumors as compared to peritumoral tissues. No association was found between GPC3 mRNA and protein expression and the clinical-pathological parameters. The Kaplan-Meier curves suggested that elevated levels of GPC3 mRNA are associated with relapse. Our results indicate differential expression of GPC3 in mammary tumors in comparasion to normal breast tissues. They also suggest the potential role of GPC3 as a biomarker and the importance of deepening the study.

Prognostic value of glypican-3 in patients with HBV-associated hepatocellular carcinoma after liver transplantation

Glypican-3 (GPC-3) is frequently overexpressed in hepatocellular carcinoma (HCC). Recent studies have shown that GPC-3 is a highly efficient diagnostic biomarker of HCC and an indicator of poor prognosis in HCC patients who have undergone hepatectomy. However, its prognostic value in patients with HBV-associated HCC after liver transplantation (LT) is not clear. The present study is to evaluate the prognostic value of GPC-3 in patients with HBV-associated HCC after LT. A cohort of 104 HCC patients with HBV-associated cirrhosis who had undergone LT at our hospital between 2002 and 2011 were enrolled in this study. Samples of HCC were taken from these patients. GPC-3 protein expression was detected in paraffin-embedded specimens using immunohistochemistry. All related clinical data were obtained from the China Liver Transplant Registry. The relationship between GPC-3 expression and clinicopathological parameters was analyzed. Univariate and multivariate Cox-regression analyses were used to identify risk factors for poor prognosis. GPC-3 was expressed in samples from 74 (71.2%) of the 104 patients. GPC-3 was expressed only in HCC cells. Positive staining was correlated with tumor size (P=0.004), encapsulation (P=0.018), pathological stage (P=0.027), portal vein invasion (P=0.043), tumor differentiation (P=0.002) and the Milan criteria (P=0.016). The 5-year survival rate and disease-free survival rate of patients with GPC-3-positive were lower than those (38.2% vs 75.4%, P<0.001; 30.8% vs 69.7%, P=0.001) of patients with GPC-3-negative. Multivariate Cox-regression analysis revealed that GPC-3 was an independent risk factor for 5-year survival rate (P=0.031) and disease-free survival rate (P=0.047), together with tumor differentiation, Milan criteria and pre-operative alpha-fetoprotein. GPC-3 is a potential biomarker for poor prognosis after LT in HCC patients with HBV-associated cirrhosis.

Differential expression of glypican-3 (GPC3) in lung squamous cell carcinoma and lung adenocarcinoma and its clinical significance.

In this study, we examined the expression of glypican-3 (GPC3) in the 2 most common histological types of lung cancer, squamous cell carcinoma and adenocarcinoma, and explored the relationship between GPC3 expression and the prognosis of these 2 types of lung cancers. Lung cancer tissues and paracancerous tissues were collected from a total of 60 patients with lung squamous cell carcinoma or lung adenocarcinoma. GPC3 gene and protein expression in the tissue samples was examined using fluorescence-based real-time quantitative polymerase chain reaction, immunohistochemistry, and western blot analysis. In addition, the serological levels of GPC3 protein in lung cancer patients were analyzed using enzyme-linked immunosorbent assays. The overall expression of GPC3 protein in lung cancer was 45% (21/60). No GPC3 expression was detected in paracancerous lung tissues. Positive expression of GPC3 protein in lung squamous cell carcinoma was significantly higher than that in lung adenocarcinoma (70 vs 20%, P < 0.001). Among GPC3-positive lung squamous cell carcinoma and lung adenocarcinoma samples, samples collected from patients with lymph node metastasis and patients with poorly differentiated cancer exhibited more pronounced GPC-3 expression. GPC3 protein expression was significantly higher in lung squamous cell carcinoma than in lung adenocarcinoma. GPC3 may be a candidate marker for detecting lung squamous cell carcinoma.

Expression of glypican 3 enriches hepatocellular carcinoma development-related genes and associates with carcinogenesis in cirrhotic livers

Glypican-3 (GPC3) protein expression was determined by immunohistochemical analysis from 29 normal livers, 80 cirrhotic livers sample taken near hepatocellular carcinoma (HCC), and 87 cirrhotic livers without HCC. The levels for miR-657 and HCC-related gene mRNAs were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Also, a published microarray dataset was used for gene set enrichment analysis (GSEA) to investigate the relationship between GPC3- and HCC-related gene signatures. Kaplan-Meier analysis was used to evaluate the relationship between GPC3 and HCC recurrence. GPC3 protein expression was not detected in any of the 29 (0%) normal livers, but was detected in 32 of 87 (37%) cirrhotic livers without HCC, and 51 of 80 (64%) cirrhotic liver samples taken near HCC sites (P < 0.001). The GPC3-positive rate in cirrhotic livers of viral origin was 68% (27/40), which was significantly higher than for non-viral cirrhotic livers (11%, 5/47) (P < 0.001). Also, GPC3 expression positively correlated with mRNA expression of HCC-related genes in the qRT-PCR and GSEA evaluations. Furthermore, HCC recurrence in cirrhotic liver samples taken near HCC sites was significantly higher in the GPC3-positive group than the GPC3-negative group (Log-rank P = 0.02, HR = 3.26; 95% CI = 1.20-10.29). This study demonstrated that highly expression of GPC3 could enrich HCC-related genes' mRNA expression and positive associate with dysplasia in cirrhotic livers. Therefore, GPC3 may serve as a precancerous biomarker in cirrhotic livers.

Glypican-3 is a potential prognostic biomarker for hepatocellular carcinoma after curative resection

Glypican-3 (GPC3), an oncofetal protein, is overexpressed in hepatocellular carcinoma (HCC). The diagnostic efficacy of GPC3 for HCC has been evaluated intensively in recent years; however, the prognostic value of GPC3 for HCC has not been well clarified. The purpose of this study was to investigate the relationship between GPC3 and postoperative patient survival in a prospective database. GPC3 protein was detected by immunohistochemistry. The relationship between GPC3 expression level and patients' clinicopathologic factors was analyzed. Kaplan-Meier survival analysis was used to calculate patients' survival and was compared by using the log rank test. The Cox regression model was used to identify the risk factors associated with prognosis. GPC3 was expressed in 84% of HCC tissues. GPC3 protein expression was correlated with thenumber of tumors (P = .015), serum alpha-fetoprotein (AFP) level (P = .011), and TNM stage (P=.006). High GPC3 expression was an independent risk factor for poor postoperative disease-free survival (hazard ratio [HR] = 0.469; 95% confidence interval [CI] 0.303-0.727; P = .001); and overall survival (HR = 0.435; 95% CI, 0.257-0.736; P = .002). Stratification analysis indicated that GPC3 had a good predictive value for tumor recurrence in patients with HCC who have normal serum AFP levels. Also, GPC3 expression status could predict the outcomes of patients with stage I disease. GPC3 is a potential and reliable biomarker for predicting tumor recurrence and overall survival in HCC patients after curative resection.

Glypican 3 expression in tumors with loss of SMARCB1/INI1 protein expression

Glypican 3 (GPC3), a membrane-bound heparin sulfate proteoglycan, is mutated in Simpson-Golabi-Behmel syndrome, characterized by tissue overgrowth and an increased risk of embryonal malignancies such as Wilms tumor. Malignant rhabdoid tumor (MRT), originally described as a rhabdomyosarcomatoid variant of Wilms tumor, is a tumor with loss of SMARCB1/INI1 protein expression. We analyzed the frequency of GPC3 protein expression, GPC3 mRNA, and serum-soluble GPC3 levels in 71 cases of tumors with loss of SMARCB1/INI1 protein expression, including 14 MRTs, 48 epithelioid sarcomas (ES) (proximal-type, 21; distal-type, 27), 4 extraskeletal myxoid chondrosarcomas, and 5 pediatric undifferentiated soft-tissue sarcomas. We found that GPC3 overexpression of more than 10% of the labeling index was recognized in 6 (42.9%) MRTs, 1 (2.1%) proximal-type ES, and 3 (60%) pediatric undifferentiated soft-tissue sarcomas (MRT vs ES, P = .0003). All the remaining cases revealed GPC3-absent expression of less than 1% of the labeling index. The median values of GPC3 mRNA in the GPC3-absent expression group and overexpression group were 10.2 and 309, respectively, with a statistically significant difference between these 2 groups (P = .004). However, there was no statistically significant difference in the prognoses of these 2 groups of MRT (P = .99). In analyzable cases of small-number MRT and pediatric undifferentiated soft-tissue sarcoma, there is no significant correlation between GPC3 immunoreactivity and serum-soluble GPC3 level. Therefore, evaluation of GPC3 immunoexpression may be a useful diagnostic tool to distinguish ES from MRT, especially extrarenal MRT. It was suggested that MRTs with GPC3 overexpression may become a new target of GPC3 immunotherapy.

Selection of small interfering RNA targeting Glypican-3 and its influence on liver cancer cells

Objective To study the action mechanism of Glypican-3 ( GPC3 ) in the development of hepatocellular carcinoma (HCC).Methods siRNA targeting GPC3 was designed and transfected into Huh7 cells,and the inhibitory effects on the expression of GPC3 was tested by using real-time polymerase chain reaction (PCR).The siRNA with obvious inhibitory effect was selected to down-regulate the GPC3 expression,and then the expression of GPC3 protein was detected by usig Wesnter blotting.The cell cycle,proliferation and invasion of Huh7 cells were assayed by using flow cytometry,fluorescence microscopy and Transwell,respectively.Results We successfully screened out a siRNA specifically targeting GPC3,its inhibition efficiency was 74.87％ and the siRNA could significantly inhibit the transcription and translation of GPC3.The knock-down of GPC3 could inhibit the proliferation and invasion of Huh7 cells,but had no effects on the cell cycle.Conclusion GPC3 expression facilitates the proliferation and invasion of HCC. Key words: Carcinoma,hepatocellular; Glypican-3; Small interfering RNA; Huh7 cells

A Hepatocellular Carcinoma Tumor Marker and Hedgehog Pathway Mediator, Glypican-3, Inhibits Hepatic Stellate Cell Viability: A Novel Outcome of Hepatic Tumor-Stroma Interactions

Introduction: Human hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide, and its incidence is rapidly increasing in the U.S. HCC is truly 2 diseases in 1 as it often arises in the setting of cirrhosis. We previously identified dysregulation of the Hedgehog (Hh) pathway as a novel mechanism for hepatocarcinogenesis and identified a role for Hh signaling in maintaining the viability and regulating the fibrinogenic capacity of hepatic stellate cells (HSC), which are implicated in cirrhosis. It is increasingly recognized that HSCs and HCCs can communicate via paracrine signaling, but Hh signaling has not been studied in this process. We hypothesized that a secreted HCC tumor marker and Hh mediator, Glypican-3 (GPC3), may play a role in regulating HCC (tumor)-HSC (stroma) interactions. Methods: We developed an in vitro co-culture (CC) model system with Transwell plates to recapitulate HCC-HSC interactions in order to assess HCC effects upon HSC viability, apoptosis, and gene transcription. We employed 2 Hh-responsive human HCC lines [PLC/PRF/5 (PLC) and SK-Hep1 (SK)] and 1 human HSC line (LX-2). Assays for cell viability (WST) and apoptosis (caspase-3/7 activity) were performed (48-72 h, N=3-9). Using real-time RT-PCR, mRNA expression of Hh target genes was studied. Western analysis was used to study GPC3 protein expression during CC. Recombinant human GPC3, as well as other Hh ligand inhibitors were analyzed for effects. Results: Using our CC system, both HCC lines had no effect upon HSC viability but caused a reduction in HSC apoptosis by 2% (SK) and 50% (PLC). This suggested that HCCs secrete paracrine factor(s) that increase HSC apoptosis and decrease HSC proliferation. Concomitantly, CC resulted in transcriptional downregulation of the Hh target gene, Gli1 (SK: 1.5-fold, PLC: 2.7-fold). at baseline, real-time RT-PCR showed that PLC expressed >73,600-fold more Gpc3 mRNA than SK. CC led to a mixed response in Gpc3 mRNA levels with a 7.3-fold decrease in PLC mRNA but 4.5-fold increase in SK mRNA. at the protein level, GPC3 was strongly expressed in both lines after CC. to determine if GPC3 affects HSCs, we treated LX-2 with GPC3 (5 μg/ml). This resulted in an 83.4% reduction in HSC viability at 48 h. Similar effects were seen with 2 other anti-Hh treatments [5E1 (anti-Hh antibody) and HIP (Hh-interacting protein)]. Conclusions: For the first time, we show that HCC-HSC interactions lead to downregulation of Hh signaling in HSC. in part, this appears to be mediated by HCC's paracrine secretion of GPC3, an HCC biomarker, which inhibits Hh signaling and HSC viability. the underlying reasons for this inhibition remain unclear but may be related to GPC3 regulation of peritumoral angiogenesis. in summary, we expand upon data suggesting a role for tumor-stroma interactions in hepatic carcinogenesis-fibrinogenesis. These findings may have clinical implications, as a trial is underway to investigate the efficacy of anti-GPC3 therapy for HCC.

Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Glypicans are heparan sulfate proteoglycans that are bound to the cell surface by glycosylphosphatidylinositol. While six members of the glypican family are known in mammals, our study focused on glypican 3 (GPC3). Loss-of-function mutations of GPC3 result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and postnatal liver and other organ overgrowth. GPC3 is overexpressed in human hepatocellular carcinoma; however, its role in normal liver regeneration and hepatocyte proliferation is unknown. Here we investigated the role of GPC3 in hepatocyte proliferation. GPC3 mRNA and protein levels begin to increase 2 days after hepatectomy with peak expression levels by day 5. In hepatocyte cultures, GPC3 reaches a plateau when hepatocyte proliferation decreases. In vitro studies using Morpholino oligonucleotides showed that blocking GPC3 expression promoted hepatocyte growth. Yeast two-hybrid assays revealed that GPC3 interacts with CD81, a member of the tetraspanin family that is reported to be involved in hepatitis C virus infection and cell proliferation. We found that CD81 levels also increased 2 days after partial hepatectomy and toward the end of regeneration. Immunofluorescence showed that CD81 and GPC3 colocalize by 2 and 6 days after hepatectomy. Co-immunoprecipitation validated the interaction of GPC3 and CD81. Our results indicate that GPC3 may be a negative regulator of liver regeneration and hepatocyte proliferation, and that this regulation may involve CD81.

Glypican‐3 protein expression in primary and metastatic melanoma

The incidence of melanoma is increasing. Fine-needle aspiration (FNA) is critical in documenting recurrent/metastatic disease in established cases. The potential of metastatic melanoma (MM) to mimic epithelial tumors presents a diagnostic dilemma. In liver FNA, the distinction between hepatocellular carcinoma (HCC) and MM is a frequent challenge. Glypican-3 (GPC3), a heparan sulfate proteoglycan, is a highly sensitive and specific marker for HCC. Serum GPC3 was shown to be expressed in 40% of primary melanomas (PMs), but to the authors' knowledge no tissue studies to date have assessed GPC3 expression in MM. In this study, GPC3 protein expression was investigated in FNAs from MM, and in corresponding histologic sections from the primary tumors. Sixty archival, direct FNA smears or CytoLyt-fixed samples from 50 patients with MM were retrieved together with formalin-fixed, paraffin-embedded specimens available from 17 corresponding PMs. All cases were stained with anti-GPC3 antibody. FNA and core biopsy specimens from HCCs and benign liver were used as positive and negative controls. GPC3 expression was divided into 2 categories: negative (negative or weak cytoplasmic staining) and positive (moderate or strong cytoplasmic with membranous accentuation). All FNAs from MM cases were negative (0 of 60) for GPC3. The exact 95% Clopper-Pearson confidence interval was 0.0% to 5.96%. Only 1 case of PM (1 of 17; 5.9%) demonstrated weak focal cytoplasmic staining (regarded as negative). In the current study, all MM and PM cases in archival FNAs and tissue sections were found to be negative for GPC3. These data suggest that GPC3 is not expressed in melanoma using the 1G12 clone.

Expression of the Glypican-3 Gene in α-fetoprotein-negative Human Hepatocellular Carcinoma

Objective: To investigate the expression of Glypican-3 gene in α-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC) and clarify whether Glypican-3 expression is a useful parameter for the diagnosis of hepatocellular carcinoma (HCC), especially for AFP-negative ones. Methods: Forty-one specimens of AFP-negative hepatocellular carcinoma and para-carcimoma tissue were studied for the expression of Glypican-3 by Northern blot. The expression of Glypican-3 protein was detected immunohistochemically with specific polyclonal antibody. Results: Northern blot analysis indicated that the expression of Glypican-3 mRNA was intensively detected in 30 of 41 AFP-negative HCC (73.17%). In contrast, Glypican- 3 mRNA was only weakly detected in 4 of the surrounding non-tumor liver tissues. There was significant difference in the Glypican-3 mRNA expression in large tumors (>5 cm) (79.31%) and in small tumors (≤5 cm) (41.67%) (P<0.01). Glypican-3 mRNA was more frequently overexpressed in poorly differentiated HCC than in well differentiated ones (76.47% vs. 42.86%, P<0.05). The Glypican-3 expression was not correlated with age, sex, HbsAg seropositivity, fibrocapsule, portal venous embolus and intrahepatic metastasis. The overexpression of Glypican-3 protein in HCC was targeted in tumor cells, not in bile duct cells and other interstitial cells. Conclusion: Glypican-3 was specially overexpressed in AFP-negative HCC, and its expression was closely correlated with the tumor size and tumor grade. Glypican-3 gene may play important roles in hepatocarcinogenesis, and can be used as a new biochemical marker of HCC, especially for AFP-negative HCC.