Glypican 3
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Glypican-3 is a protein that, in humans, is encoded by the GPC3 gene. The GPC3 gene is located on human X chromosome (Xq26) where the most common gene (Isoform 2, GenBank Accession No.: NP_004475) encodes a 70-kDa core protein with 580 amino acids. Three variants have been detected that encode alternatively spliced forms termed Isoforms 1 (NP_001158089), Isoform 3 (NP_001158090) and Isoform 4 (NP_001158091).
Structure
The protein core of GPC3 consists of two subunits, with an N-terminal subunit of approximately 40 kDa and a C-terminal subunit of approximately 30 kDa.
GPC3 belongs to the glypican family, one of six glypicans (GPC1–6) identified in mammals. Glypicans are cell surface heparan sulfate proteoglycans composed of a membrane-associated protein core substituted with a variable number of heparan sulfate chains. Members of the glypican-related integral membrane proteoglycan family (GRIPS) are anchored to the plasma membrane through a glycosyl phosphatidylinositol linkage.
A cysteine-rich domain within the N-terminal lobe of GPC3 forms a hydrophobic groove that binds Wnt3a. In addition to protein interactions, Wnt recognizes specific heparan sulfate structures on GPC3 containing IdoA2S and GlcNS6S, while 3-O-sulfation of GlcNS6S3S further enhances Wnt binding.
Function
GPC3 regulates multiple signaling pathways involved in cell growth and development, including Wnt/β-catenin and Yap signaling. As a member of the glypican family, GPC3 may contribute to the control of cell division and growth regulation.
In the Wnt pathway, GPC3 interacts with both Wnt ligands and frizzled (FZD) receptors to form a signaling complex that activates downstream signaling. The GPC3 core protein is thought to function as a co-receptor or receptor for Wnt ligands. Blocking the Wnt-binding domain of GPC3 with the HN3 single-domain antibody inhibits Wnt activation.
GPC3 also modulates Yap signaling through interaction with FAT1, a potential upstream cell surface receptor of YAP1 in human cells.
In addition, GPC3 has been reported to bind Alpha-fetoprotein in liver cancer.
Clinical signficance
Disease linkage
Deletion mutations in this gene are associated with Simpson–Golabi–Behmel syndrome.
Diagnostic utility
Glypican 3 immunostaining has utility for differentiating hepatocellular carcinoma (HCC) and dysplastic changes in cirrhotic livers; HCC stains with glypican 3, while liver with dysplastic changes and/or cirrhotic changes does not. Using the YP7 murine monoclonal antibody, GPC3 protein expression is found in HCC, not in normal liver and cholangiocarcinoma. The YP7 murine antibody has been humanized and named as 'hYP7'. GPC3 is also expressed to a lesser degree in melanoma, ovarian clear-cell carcinomas, yolk sac tumors, neuroblastoma, hepatoblastoma, Wilms' tumor cells, and other tumors. However, the significance of GPC3 as a diagnostic tool for human tumors other than HCC is unclear.
Therapeutic potential
To validate GPC3 as a therapeutic target in liver cancer, the anti-GPC3 therapeutic antibodies GC33, YP7, HN3 and HS20 have been made and widely tested. The laboratory of Dr. Mitchell Ho at the National Cancer Institute, NIH (Bethesda, Maryland, US) has generated YP7 murine monoclonal antibody that recognizes the C-lobe of GPC3 by hybridoma technology. The antibody has been humanized (named hYP7) via antibody engineering for clinical applications. The Ho lab has also identified the human single-domain antibody ('human nanobody') HN3 targeting the N-lobe of GPC3 and the human monoclonal antibody HS20 targeting the heparan sulfate chains on GPC3 by phage display technology. Both HN3 and HS20 antibodies inhibit Wnt signaling in liver cancer cells . The immunotoxins based on HN3, the antibody-drug conjugates based on hYP7 and the T-cell engaging bispecific antibodies derived from YP7 and GC33, have been developed for treating liver cancer. The chimeric antigen receptor (CAR) T cell immunotherapies based on GC33, hYP7 and HN3 are being reported at various stages for treating liver cancer. In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells can eliminate GPC3-positive cancer cells, by inducing perforin- and granzyme-mediated cell death and reducing Wnt signaling in tumor cells. CAR (hYP7) T cells are being evaluated at a clinical trial at the NIH.