Author Affiliations: Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, China (Wang L, Pan LH, Qian Q and Yao DF); Departments of Medical Informatics (Wang L) and Immunology (Yao M), Medical School of Nantong University, Nantong 226001, China

Corresponding Author: Deng-Fu Yao, MD, PhD, Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, 20 West Temple Road, Nantong 226001, China (Tel: +86-513-85052297; Fax: +86- 513-85052254; Email: yaodf@ahnmc.com)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60396-4

Published online July 2, 2015.

 

 

Contributors: WL, YM, PLH and QQ wrote the manuscript. YDF is the guarantor.

Funding: The study was supported in part by the grants from the Projects of Jiangsu Medical Science (2013-WSW-011, 2014-YY-028 and HK201102), the Qinglan and PAPD of Jiangsu Higher Education, the Nantong Undertaking and Technological Innovation (H2014078), and the International S&T Cooperation Program (2013DFA32150) of China.

Ethical approval: Not needed.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multi-step and complex process. Early diagnosis and effective treatments are of utmost importance. This review summarized the recent studies of oncofetal glypican-3 (GPC-3), a membrane-associated heparan sulfate proteoglycan, in the diagnosis and treatment of HCC.

 

DATA SOURCES: English-language reports published from June 2001 to September 2014 were searched from MEDLINE. The key words searched included: GPC-3, biomarker, target and HCC. The sensitivity, specificity, positive and negative predictive values were extracted, and the effect of GPC-3 targeted therapy on HCC was also evaluated.

 

RESULTS: GPC-3 plays a crucial role in HCC cell proliferation and metastasis. It mediates oncogenesis involving signaling pathways during hepatocyte malignant transformation. GPC-3 expression is increased in atypical hyperplasia and cancerous tissues. GPC-3 levels in HCC patients are related to HBV infection, TNM stage, periportal cancerous embolus, and extrahepatic metastasis. The diagnostic accuracy of the combination of serum GPC-3 and alpha-fetoprotein in HCC is up to 94.3%. Down-regulation of GPC-3 with specific siRNA or anti-GPC-3 antibody alters cell migration, metastasis and invasion behaviors. The nude mice xenograft tumor growth is inhibited by silencing GPC-3 gene transcription.

 

CONCLUSION: Oncofetal GPC-3 is a highly specific biomarker for the diagnosis of HCC and a promising target molecule for HCC gene therapy.

 

(Hepatobiliary Pancreat Dis Int 2015;14:361-366)

 

KEY WORDS: hepatocellular carcinoma; glypican-3; signal pathways; diagnosis; targeted therapy

Hepatocellular carcinoma (HCC) is one of the most common and fatal malignancies worldwide. HCC is associated with chronic persistent infection of hepatitis B or C virus (HBV or HCV). Other etiologies include alcohol, chemicals and aflatoxin B1.^[1-3] Most HCC patients are usually asymptomatic until later stages and the prognosis is dismal. Because HCC is not sensitive to radiotherapy or chemotherapy, liver resection or transplantation is the only potential curative treatment. The high rate of recurrence after surgery and metastasis lead to a poor prognosis with considerably lower 5-year survival rates. Therefore, the improvement of early diagnosis is essential to the successful treatment.^[4-6]

 

Glypican (GPC) is a family with six subtypes (GPC-1 to GPC-6) of heparan sulfate proteoglycans that are linked to the exocytoplasmic surface of the plasma membrane by a glycosyl-phosphatidylinositol anchor.^[7] GPC-3 is a molecule that links to cell membrane surface with stronger immunostaining, making it a useful biomarker for HCC diagnosis.^{[8, 9]} GPC-3 is associated with HCC development and progression; GPC-3 is increased in patients with HCC compared with those with pre-neoplastic lesions. Interestingly, the levels of GPC-3 mRNA are more frequently elevated than those of alpha-fetoprotein (AFP) in patients with HCC; this difference is even more obvious in patients with small-size HCC. Recently, GPC-3 was explored as one of the tumor-specific targets. Anti-GPC-3 antibody, T-cell-directed immunotherapies, and down-regulation of GPC-3 gene transcription represent alternative approaches for HCC therapy.^[10-12] The present review focused on GPC-3 as a specific biomarker for HCC diagnosis and a new target for HCC therapy.

 

 

All glypicans share a structure characterized by a conserved pattern of 14 cysteine residues which may form intra-molecular disulphide linkages.^{[7, 13, 14]} GPC-3 belongs to the glypican family, which plays an important role in cellular growth, differentiation and migration.^[15-17] Its gene is located on human X chromosome (Xq26) encoding a 70 kDa core protein which can be cleaved by furin to generate a 40 kDa N-terminal protein and a 30 kDa C-terminal protein containing two heparan sulfate glycan chains. The molecule of GPC-3 links to the cellular membrane through a glycosyl-phosphatidylinositol anchor and plays an important role in regulating cells growth.^{[18, 19]} The two subunits are produced by cleavage between Arg³⁵⁸ and Ser³⁵⁹, the cleavage generates a soluble N-terminal and a combined with membrane C-terminal- fragment. In addition, the location of hydrosulfite chains in the C-terminal region near cell membrane is conserved in all glypicans. Serine⁵⁶⁰ is predicted as a cleavage site in GPC-3 binding Wnt,^{[20, 21]} Hedgehog and fibroblast growth factor (FGF)-2 through its core protein and/or the hydrosulfite chains. GPC-3 contributes to cell migration, invasion, angiogenesis and apoptosis, possibly through its interactions with the Wnt and Hedgehog pathways,^{[22, 23]} or modulates FGF-2 and bone morphogenetic protein-7 signaling.^[24] GPC-3 is not expressed in normal adult liver.

 

 

The Wnt signaling pathway is essential in many biological processes. The biological role of the interaction between Wnt and GPC-3 remains to be elucidated. The dynamic alterations of rat GPC-3 and GPC-3 mRNA were investigated in hepatocarcinogenesis, showing that GPC-3 is of diagnostic value at early stage of HCC.^[25-28] Positive GPC-3 staining was located in hepatocyte cytoplasm at morphological stages of granule-like degeneration, atypical hyperplasia (precancerous), and cancer formation, with a progressive increase of liver RNA level and gamma-glutamyl transpeptidase activity, indicating that GPC-3 expression is associated with hepatocyte malignant transformation. Hepatocyte oncogenesis could be induced by GPC-3 through insulin-like growth factor II (IGF-II) pathway activation, by zinc fingers and homeoboxes 2 (Zhx2) regulation or by AFP regulator 2 (Afr2) in regenerating the liver.^{[29, 30]}

 

HCC is characterized by poor prognosis and hard to be early diagnosed. The positive rate of liver GPC-3 mRNA, liver GPC-3, and circulating GPC-3 were 100%, 100% and 77.8% in HCC, 100%, 100%, and 66.7% in precancerous patients, 83.3%, 83.3%, and 38.9% in degeneration group and negative in livers or blood of controls, respectively. There was a close positive correlation between liver GPC-3 mRNA and total RNA level (r=0.475, P<0.05) or liver GPC-3 protein (r=1.0, P<0.001) or serum GPC-3 (r=0.994, P<0.001). The mechanism of oncogenic GPC-3 activation in human HCC involves reduced nucleic Zhx2 or by c-Myc gene, indicating that the abnormal expressions of circulating GPC-3 and GPC-3 mRNA in hepatocarcinogenesis may be early molecular markers for HCC diagnosis.^{[25, 28]}

 

 

Oncofetal GPC-3 expression was found in cytoplasm and cell membrane in most (70% to 100%) of HCC tissues (Table 1). Yao and colleagues^[37] found that the positive rate of GPC-3 was 80.6% in HCC patients, 41.7% in their paracancerous tissues and no expression in their distal cancerous tissues. The intensity of GPC-3 in HCC is significantly higher than that in their surrounding tissues. GPC-3 is a developmentally-regulated oncofetal protein that is a clinically-relevant biomarker for early HCC diagnosis and one of the first transcripts to appear during the hepatocyte malignant transformation; about 50% of high-grade dysplastic macronodules in cirrhotic liver have GPC-3 expression.^[41]

 

To identify a molecule signature for early HCC, Llovet and coworkers^[42] analyzed 55 genes and found that GPC-3 is positive in all HCC and negative in all of the dysplastic nodules, suggesting that GPC-3 is a specific biomarker for HCC diagnosis. Hepatic fine needle aspirates showed that GPC-3 immunoreactivity was 83% to 90% of HCC cases, whereas all benign lesions and metastatic carcinomas were nonreactive.^[36] Diagnostic utility of GPC-3 in the specimens to aid in distinction of HCC from metastatic tumors and benign liver lesions, suggesting that up-regulation of circulating GPC-3 mRNA is a more sensitive and specific biomarker for monitoring metastasis of HCC.^[43]

 

 

AFP is a useful marker for HCC diagnosis and for the evaluations of therapeutic strategy. However, AFP is also positive in benign liver diseases which make the differential diagnosis very difficult. Circulating GPC-3 is increased in patients with HCC which implies that GPC-3 may be very valuable in HCC diagnosis and monitoring the progress. Currently, many biomarkers have been applied in clinical practice in HCC diagnosis. However, only a few markers such as HS-GGT,^[44] AFP-L3^[45] are sensitive with reasonable specificity. A recent study^[37] demonstrated that blood GPC-3 is superior to AFP in specificity, positive and negative predictive value, and accuracy in HCC diagnosis. Furthermore, GPC-3 is also superior to AFP in therapeutic decision making and predicting the prognosis of HCC patients.

 

Serum GPC-3 is detectable in 52.8% of HCC patients, with a specificity of 97.1%. The serum GPC-3 is detected in only 1.4% to 2.0% of patients with other liver disease.^[37] In comparison, AFP is not accurate for HCC diagnosis because of its higher false positive rate (14.3% to 35.0%) in benign liver diseases. The sensitivity of AFP-L3 is 53.3% and the specificity, 88.9%.^[45] Serum GPC-3 could be used to differentiate HCC from non-malignant chronic liver disease and other liver cancers. However, the combination of circulating GPC-3, GPC-3 mRNA and AFP significantly improves the accuracy of HCC diagnosis.^[37]

 

 

The prognosis of patients with HCC is poor because of the later diagnosis and high recurrence after surgery.^[2,6] Metastasis is the final stage in HCC progression and is thought to be responsible for up to 90% of HCC deaths.^{[46, 47]} GPC-3 gene transcription from liver tissues and circulating peripheral blood mononuclear cells is associated with extrahepatic metastasis of HCC. In a study, GPC-3 mRNA was expressed in 143 out of 191 (74.9%) primary and recurrent HCC, but in only 5 of 154 (3.2%) normal livers, indicating that expression of GPC-3 mRNA is low or absent in normal liver, focal nodular hyperplasia and liver cirrhosis. GPC-3 gene fragments are detectable by DNA sequencing in most cancerous tissues or circulating peripheral blood mononuclear cells from HCC patients, but not in distal cancerous liver tissues or cells from benign liver diseases.^{[48, 49]}

 

Circulating levels of GPC-3 mRNA are related to TNM stage, periportal cancerous embolus, and extrahepatic metastasis. Interestingly, GPC-3 has a high incidence in early and small HCC in patients with periportal cancer embolus (100%) and in those with extrahepatic metastasis (100%).^{[37, 50]}

 

 

GPC-3 overexpression plays an important role in HCC transformation, proliferation and metastasis. Therefore, GPC-3 is a molecular target for HCC therapy.^{[51, 52]} Silencing GPC-3 gene transcription by specific shRNA inhibits HCC cell proliferation, with 71.1% inhibited in shRNA group, and 80.1% in shRNA plus sorafenib (100 µmol/L) group. A total of 65.6% of HCC cells were arrested in G1 phase. Cell apoptosis was significantly increased to 66.8% in contrast to 6.92% in the neg-shRNA group.^[53] GPC-3 as a target for HCC therapy has been investigated with GPC-3 vaccine and anti-GPC-3 antibodies. Some advances in GPC-3 use as a new target for HCC therapy are summarized in Table 2. Among them, antibody-based therapy is the most outstanding clinically. Given that GPC-3 is increased in early, high-grade dysplastic macronodules, plus that the significant proportion of overt HCC are immunoreactive with anti-GPC-3,^[54] a therapeutic mAb (GC33, aa524-563) in the C-terminal portion of GPC-3 is generated in MRL/lpr mice against a GST-fusion. The antibody causes cytotoxicity; however it is effective in the xenograft model.^{[55, 56]}

 

The xenograft model is commonly used in tumor studies because of its convenient operation, high success rate, short latent period, and easy to monitor. Besides anti-GPC-3 antibody, miRNA targeted for GPC-3 also has therapeutic effect on HCC model. Tumor formation and growth are significantly inhibited by miRNA in HepG2 cell induced HCC model in nude mice compared with the same model without miRNA treatment.^[60] Immunohistochemical analysis confirmed that down-regulating GPC-3 with miRNA significantly decreases the β-catenin, p-GSK3β, and cyclin D1 expressions. It is well known that β-catenin and GSK3β not only play important roles in regulating metabolism, transcription, embryonic development, and other processes, but also serve the key role in the Wnt/β-catenin induced GSK3β phosphorylation which results in the dissolution of the complex responsible for the degradation of β-catenin.^[61,62] Targeting GPC-3 for therapeutic intervention is a promising approach for the clinical management of HCC.^[63,64] Soluble GPC-3 significantly inhibits the growth of HCC both in vitro and in vivo. Analysis of soluble GPC3 expressing tissues showed that the secreted glypican inhibits angiogenesis and HCC growth by acting at levels of the pro-tumorigenic phenotype.^[65]

 

 

HCC is a highly chemoresistant cancer with no effective medication. Molecular targeted therapy offers an optimal option for HCC patient who cannot be treated by surgery. However, molecular therapy remains a challenge mainly due to lack of specific targets. Oncofetal GPC-3 promotes HCC development through up-regulation of the Wnt/β-catenin pathway that provides a novel therapy for HCC. GPC-3 is not only a promising biomarker but also a therapeutic target for HCC.^[66] Further study should be conducted in combination of specific siRNA and multi-targets for HCC therapy.

 

 

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