Mariana Capurro

Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma.

BACKGROUND & AIMS Early detection of hepatocellular carcinoma (HCC) is critical for successful treatment. However, the differential diagnosis between HCC and benign hepatic lesions is sometimes difficult and new biochemical markers for HCC are required. It has been reported that glypican-3 (GPC3) messenger RNA (mRNA) is significantly increased in most HCCs compared with benign liver lesions or normal liver. The goal of this study is to determine whether GPC3 is also overexpressed at the protein level and whether GPC3 is detectable in the serum of patients with HCC. METHODS GPC3 was assessed in liver tissue sections by immunohistochemistry and in serum by enzyme-linked immunosorbent assay. Serum alpha-fetoprotein (AFP) level was also measured in the same patients. RESULTS Immunohistochemical studies showed that GPC3 is expressed in 72% of HCCs (21 of 29), whereas it is not detectable in hepatocytes from normal liver and benign liver diseases. Consistent with this, GPC3 was undetectable in the serum of healthy donors and patients with hepatitis, but its levels were significantly increased in 18 of 34 patients (53%) with HCC. In addition, only 1 of 20 patients with hepatitis plus liver cirrhosis displayed elevated levels of serum GPC3. Interestingly, in most cases, there was no correlation between GPC3 and AFP values. Thus, at least 1 of the 2 markers was elevated in 82% of the patients with HCC. CONCLUSIONS GPC3 is specifically overexpressed in most HCCs and is elevated in the serum of a large proportion of patients with HCC. The simultaneous determination of GPC3 and AFP may significantly increase the sensitivity for diagnosis of HCC.

Glypican-3 Promotes the Growth of Hepatocellular Carcinoma by Stimulating Canonical Wnt Signaling

Glypican-3 (GPC3) is a heparan sulfate proteoglycan that is bound to the cell membrane by a glycosyl-phosphatidylinositol anchor. GPC3 is expressed by most hepatocellular carcinomas but not by normal hepatocytes and benign liver lesions. We report here that GPC3 stimulates the in vitro and in vivo growth of hepatocellular carcinoma cells by increasing autocrine/paracrine canonical Wnt signaling. Co-immunoprecipitation experiments showed that GPC3 is able to form complexes with Wnts, and cell-binding assays indicated that GPC3-expressing cells have an increased capacity to bind Wnt. Collectively, these results suggest that GPC3 stimulates Wnt activity by facilitating the interaction of this polypeptide with its signaling receptors. Surprisingly, in contrast to the current model that proposes that Wnt-glypican binding is mediated by the heparan sulfate chains, we found that the nonglycanated GPC3 core protein can form complexes with Wnts. Furthermore, we showed that the glycosaminoglycan chains are not required for the stimulatory effect on Wnt signaling and hepatocellular carcinoma growth.

Glypican-3 Inhibits Hedgehog Signaling during Development by Competing with Patched for Hedgehog Binding

Loss-of-function mutations in glypican-3 (GPC3), one of the six mammalian glypicans, causes the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and GPC3 null mice display developmental overgrowth. Because the Hedgehog signaling pathway positively regulates body size, we hypothesized that GPC3 acts as an inhibitor of Hedgehog activity during development. Here, we show that GPC3 null embryos display increased Hedgehog signaling and that GPC3 inhibits Hedgehog activity in cultured mouse embryonic fibroblasts. In addition, we report that GPC3 interacts with high affinity with Hedgehog but not with its receptor, Patched, and that GPC3 competes with Patched for Hedgehog binding. Furthermore, GPC3 induces Hedgehog endocytosis and degradation. Surprisingly, the heparan sulfate chains of GPC3 are not required for its interaction with Hedgehog. We conclude that GPC3 acts as a negative regulator of Hedgehog signaling during mammalian development and that the overgrowth observed in SGBS patients is, at least in part, the consequence of hyperactivation of the Hedgehog signaling pathway.

Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling

Binding between the Hedgehog ligand and its receptor Patched 1 is stabilized by Glypican-5.

Glypican-3: a marker and a therapeutic target in hepatocellular carcinoma

Glypican‐3 (GPC3) is a member of the glypican family. Glypicans are proteoglycans that are attached to the cell surface by a glycosyl‐phosphatidylinositol anchor. They regulate the signaling activity of several growth factors, including Wnts. This regulation is based on the ability of glypicans to stimulate or inhibit the interaction of these growth factors with their respective signaling receptors. It has been clearly established that whereas GPC3 is expressed by most hepatocellular carcinomas (HCCs), this glypican is not detected in normal and cirrhotic liver, or in benign hepatic lesions. Consequently, immunostaining of liver biopsies for GPC3 is currently being used by clinical pathologists to confirm HCC diagnosis when the malignant nature of the lesion is difficult to establish. In addition to being a marker of HCC, GPC3 plays a role in the progression of the disease. GPC3 promotes the growth of HCC by stimulating canonical Wnt signaling. It has been proposed that this stimulation is based on the ability of GPC3 to increase the binding of Wnt to its signaling receptor, Frizzled. Two therapeutic approaches for HCC that target GPC3 are currently being tested in phase II clinical trials. One of them is based on the use of a humanized GPC3 monoclonal antibody that inhibits the in vivo growth of HCC xenografts by inducing antibody‐dependent cellular cytotoxicity. The second approach employs a vaccine that consists of two GPC3‐derived peptides that induce cytotoxic T lymphocytes against these peptides. Targeting of GPC3 might offer a new tool for the treatment of HCC.

Soluble glypican 3 inhibits the growth of hepatocellular carcinoma in vitro and in vivo

The heterogeneity of the molecular pathology of HCC poses a formidable obstacle to the development of non‐cytotoxic therapies. Several pro‐tumorigenic signaling pathways can be aberrantly activated in HCC, including those triggered by Wnts. Glypican‐3 (GPC3), a membrane‐bound heparan sulfate proteoglycan that is overexpressed in most HCCs, promotes the growth of these tumors by stimulating Wnt signaling. Because GPC3 binds with high affinity to Wnts, and its growth‐promoting activity requires attachment to the cell membrane, we have hypothesized that a mutated GPC3 lacking the GPI anchoring domain (sGPC3) will block Wnt signaling and inhibit the growth of Wnt‐dependent tumors. In addition, because sGPC3 displays heparan sulfate chains, this secreted glypican could also inhibit HCC growth by blocking the activity of other heparin‐binding growth factors. To test this hypothesis, HCC cell lines were infected with an sGPC3‐expressing lentivirus or virus control, and the effect of sGPC3 on the in vitro and in vivo growth was investigated. In addition, the signaling pathways targeted by sGPC3 were identified. We observed that sGPC3‐expressing cells had lower proliferation rate. In addition, sGPC3 significantly inhibited the in vivo growth of the Huh6, HepG2 and Huh7 HCC cell lines. sGPC3 blocked Wnt signaling in Huh6‐ and Huh7‐derived tumors and Erk1/2 and Akt phosphorylation in tumors generated by Huh7 and HepG2 cells, respectively. An anti‐angiogenic effect in Huh7 and HepG2‐derived tumors was also observed. We conclude that sGPC3 can inhibit HCC tumorigenicity by blocking the activity of several pro‐tumorigenic growth factors.

The role of glypican-3 in the regulation of body size and cancer

Glypicans are a family of heparan sulfate proteoglycans whose members are bound to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Loss-of-function mutations in GPC3, one of the six mammalian glypicans, causes the Simson-Golabi-Behmel Syndrome. This is a disorder characterized by pre- and post-natal overgrowth, a broad spectrum of visceral and skeletal abnormalities, and an increased risk for the development of embryonic tumors. GPC3-null mice also display significant overgrowth. We have recently reported that GPC3 acts as a negative regulator of Hedgehog signaling during development, and that the overgrowth caused by the lack of functional GPC3 is due, at least in part, to the hyperactivation of Hedgehog signaling. Here we discuss the rationale that led us to hypothesize that GPC3 could be a negative regulator of Hedgehog signaling, and speculate about the implications of our discovery regarding the role of GPC3 in some cancer types. We also discuss our recent results of experiments that investigated the role of the core protein, the heparan sulfate chains, and the GPI anchor in GPC3 function. Finally, we propose an explanation for the tissue-specific function of GPC3.

The role of glypicans in Hedgehog signaling

Glypicans (GPCs) are a family of proteoglycans that are bound to the cell surface by a glycosylphosphatidylinositol anchor. Six glypicans have been found in the mammalian genome (GPC1 to GPC6). GPCs regulate several signaling pathways, including the pathway triggered by Hedgehogs (Hhs). This regulation, which could be stimulatory or inhibitory, occurs at the signal reception level. In addition, GPCs have been shown to be involved in the formation of Hh gradients in the imaginal wing disks in Drosophila. In this review we will discuss the role of various glypicans in specific developmental events in the embryo that are regulated by Hh signaling. In addition, we will discuss the mechanism by which loss-of-function GPC3 mutations alter Hh signaling in the Simpson-Golabi-Behmel overgrowth syndrome, and the molecular basis of the GPC5-induced stimulation of Hh signaling and tumor progression in rhabdomyosarcomas.

Glypican-3 binds to Frizzled and plays a direct role in the stimulation of canonical Wnt signaling

ABSTRACT Glypican-3 (GPC3) is a proteoglycan that is bound to the cell surface. It is expressed by most hepatocellular carcinomas (HCCs) but not by normal hepatocytes. GPC3 stimulates HCC growth by promoting canonical Wnt signaling. Because glypicans interact with Wnts, it has been proposed that these proteoglycans stimulate signaling by increasing the amount of Wnt at the cell membrane, thus facilitating the interaction of this growth factor with its signaling receptor, Frizzled. However, in this study, we demonstrate that GPC3 plays a more direct role in the stimulation of Wnt signaling. Specifically, we show that, in addition to interacting with Wnt, GPC3 and Frizzled interact directly through the glycosaminoglycan chains of GPC3, indicating that this glypican stimulates the formation of signaling complexes between Wnt and Frizzled. Consistent with this, we show that the binding of Wnt at the cell membrane triggers the endocytosis of a complex that includes Wnt, Frizzled and GPC3. Additional support for our model is provided by the finding that glypican-6 (GPC6) inhibits canonical Wnt signaling, despite the fact that it binds to Wnt at the cell membrane.

Processing by Convertases Is Not Required for Glypican-3-induced Stimulation of Hepatocellular Carcinoma Growth

Glypicans are a family of heparan sulfate proteoglycans that are bound to the cell surface by a lipid anchor. Six members of this family have been identified in mammals (GPC1-GPC6). Glypicans act as regulators of the activity of various cytokines, including Wnts, Hedgehogs, and bone morphogenetic proteins. It has been reported that processing by a convertase is required for GPC3 activity during convergent extension in zebrafish embryos, for GPC3-induced regulation of Wnt signaling, and for the binding of GPC3 to Wnt5a. In our laboratory, we have recently demonstrated that GPC3 promotes the growth of hepatocellular carcinomas (HCCs) by stimulating canonical Wnt signaling. Because there is increasing evidence indicating that the structural requirements for GPC3 activity are cell type specific, we decided to investigate whether GPC3 needs to be processed by convertases to stimulate cell proliferation and Wnt signaling in HCC cells. We report here that a mutant GPC3 that cannot be processed by convertases is still able to play its stimulatory role in Wnt activity and HCC growth.