The Role of Glypicans in Cancer Progression and Therapy

Abstract

Glypicans are a family of heparan sulfate proteoglycans that are attached to the cell membrane via a glycosylphosphatidylinositol anchor. Glypicans interact with multiple ligands, including morphogens, growth factors, chemokines, ligands, receptors, and components of the extracellular matrix through their heparan sulfate chains and core protein. Therefore, glypicans can function as coreceptors to regulate cell proliferation, cell motility, and morphogenesis. In addition, some glypicans are abnormally expressed in cancers, possibly involved in tumorigenesis, and have the potential to be cancer-specific biomarkers. Here, we provide a brief review focusing on the expression of glypicans in various cancers and their potential to be targets for cancer therapy.

Keywords

biomarker cancer extracellular matrix glycosaminoglycan glypican heparan sulfate proteoglycan immunostaining

Introduction

As one of the leading causes of human death, cancer remains a major health problem worldwide.¹ An understanding of molecular changes in cancer development is important to reveal new biomarkers and targets for diagnosis and treatment of the disease. Heparan sulfate proteoglycans (HSPGs), which are mainly found at the cell surface and in the extracellular matrix, have gained increased scientific interest due to their roles in malignant transformation.^2,3

Glypicans, one of the HSPG families, are comprised of three key features: a 60- to 70-kDa core protein, heparan sulfate (HS) chains, and a glycosylphosphatidylinositol (GPI) linkage that anchors glypicans to the cell surface.⁴ The core protein is predicted to form a conserved tertiary structure due to seven intramolecular disulfide bridges caused by 14 conserved cysteine residues. There are six glypican family members in humans, referred to as glypican-1 (GPC1) through glypican-6 (GPC6). In addition to the difference in the primary sequences of their protein cores, they have various numbers of HS chains. Glypicans play important roles in development by modulating morphogen gradient formation⁵ and cell growth by regulating Wnt^6–8 and other signaling pathways. Glypicans are abnormally expressed in multiples types of cancer and are crucial for cancer cell growth and progression.^9–11 For example, GPC2^12,13 and GPC3^14,15 have highly specific expression in pediatric cancers and hepatocellular carcinoma (HCC), respectively. In particular, GPC1, GPC2, and GPC3 have been studied as potential diagnostic tools and therapeutic targets. This review will summarize the latest findings related to the expression and functions of glypicans in cancers and will emphasize their potentials as emerging biomarkers and promising therapeutic targets to fight cancer.

GPC1

The GPC1 gene on chromosome 2q37 encodes the GPC1 protein, consisting of a 558-amino-acid core protein and three predicted HS chains attached at S486, S488, and S490 (UniProtKB/Swiss-Prot P35052). GPC1 has both a membrane-anchored form and a secreted soluble form.¹⁶ It is mainly expressed in the neural and skeletal systems during embryonic development and is expressed at low levels in adult tissues.¹⁷ GPC1 is involved in cancer development through interactions with growth factors such as fibroblast growth factor-2 (FGF-2),^18,19 vascular endothelial growth factor (VEGF),^20,21 and transforming growth factor beta (TGF-β).²²

GPC1 Expression in Cancer

Many studies have shown that GPC1 is upregulated in a variety of human cancers. In 1998, Kleeff et al.²³ reported that GPC1 was strongly expressed in human pancreatic cancer, by both the cancer cells and the adjacent fibroblasts, whereas its expression was low in normal pancreas and in chronic pancreatitis. Furthermore, high GPC1 expression is significantly related to the perineural invasion of pancreatic cancer and is associated with poor patient prognosis.²⁴ They also observed strong GPC1 immunoreactivity in the fibroblasts surrounding the cancer cells. More recently, Lu et al.²⁵ systematically evaluated GPC1 expression in large-scale clinical samples and detected GPC1 protein in 59.7% (111/186) of pancreatic tumor tissues. However, GPC1 protein was rarely found in normal and adjacent non-cancerous pancreas. They also reported that GPC1 was elevated in 63.6% (34/55) of early-stage cases of pancreatic cancer.

We isolated a high-affinity monoclonal antibody (MAb), HM2, that recognizes the C-lobe of GPC1 by mouse hybridoma technology.²⁶ The HM2 antibody was used to detect GPC1 protein expression in tissues by immunohistochemistry (IHC). As shown in Fig. 1, the GPC1 protein is highly expressed in pancreatic tumor tissue compared with normal pancreas, which is consistent with reports described above. Interestingly, we also detected GPC1 expression in surrounding fibroblasts, which may promote tumor metastasis and angiogenesis by interacting with growth factors.

Figure 1.

The protein expression of GPC1, GPC2, and GPC3 in cancers. GPC1 protein level is increased in PDAC tissue compared with normal pancreas as determined by IHC using the GPC1-specific mouse monoclonal antibody (MAb) HM2. GPC2 protein is presented in NB tissue but not in normal peripheral nerve tissue as determined by IHC using the anti-GPC2 mouse MAb CT3. GPC3 is overexpressed in HCC tissue compared with normal liver tissue using the GPC3-specific mouse MAb YP7 by IHC. Images were obtained under 20 × magnification. Scale bar = 200 µm. Abbreviations: HCC, hepatocellular carcinoma; IHC, immunohistochemistry; NB, neuroblastoma; PDAC, pancreatic ductal adenocarcinoma.

The overexpression of GPC1 has also been described in other cancer types, including breast cancer²⁷ and glioma,²⁸ and is associated with a poor prognosis in glioblastomas.²⁹ Suhovskih et al.³⁰ showed that GPC1 localization was altered, from the epithelial cells in normal prostate tissues, to the stroma surrounding prostate tumors. A recent report demonstrated that the expression of GPC1 and α3(V) collagen, an important factor for mammary tumor progression, was tightly associated in breast cancer.³¹ These findings suggest that the protumor roles of GPC1 may be mediated by interaction with tumor microenvironment in certain types of cancer. Moreover, 98.8% of esophageal squamous cell carcinoma (ESCC) tissues demonstrated an elevated expression of GPC1 in the IHC assessment of 175 ESCC patients, and the overexpression of GPC1 is associated with poor prognosis and chemoresistance in ESCC.³² In addition, Matsuzaki et al.³³ detected high levels of GPC1 in nearly 50% of uterine cervical cancer cases.

Multiple studies have been conducted to reveal the underlying mechanisms of upregulation of GPC1 in various cancers. Tanaka et al.³⁴ reported that KRAS and ecotropic viral integration site 1 (EVI1), two known drivers of pancreatic carcinogenesis, positively regulated GPC1 expression. Another possible mechanism is the promoter hypomethylation in the GPC1 gene in pancreatic cancer, as the methylation status of GPC1 was inversely correlated with its mRNA levels in pancreatic tumor tissues, which explains the reexpression of GPC1 in pancreatic cancer.²⁵

In addition to its membrane-anchored form, GPC1 also has a secreted soluble form and can be detected in the peripheral blood system. Several studies have been carried out to explore its potential as a clinical biomarker. In 2015, Melo et al.³⁵ first reported that GPC1 was enriched in cancer cell–derived exosomes and detected in the early stages of pancreatic cancer with 100% sensitivity and specificity. Nevertheless, some recent studies yielded controversial results. They found that high levels of serum GPC1 may be able to determine pancreatic tumor size and disease burden, but were unable to distinguish pancreatic cancer from benign pancreatic disease.^36,37 The discrepancies might originate from different technical approaches to measure exosomal GPC1 levels. Therefore, more thorough studies will be necessary to establish whether GPC1 in the blood can be a biomarker for pancreatic cancer and other cancers.

GPC1 Signaling in Cancer

One of the altered cellular activities induced by GPC1 might be through its modulation of the FGF-2 signaling pathway, which is known to regulate cell growth and survival.³⁸ Qiao et al.¹⁸ showed that GPC1 expression enhanced the growth of brain endothelial cells by promoting FGF-2 signaling. GPC1 also modulates VEGF signaling, a key factor for angiogenesis.³⁹ Whipple et al.²¹ generated a GPC1 null mouse model of pancreatic cancer and found these mice exhibited suppressed angiogenesis in conjunction with the decreased mRNA expression of VEGF-A. In addition, GPC1 is reported to modulate the TGF-β signaling pathway. Downregulation of GPC1 attenuated TGF-β1-induced cell growth inhibition and Smad 2 phosphorylation in pancreatic cancer cells.²² Kayed et al.⁴⁰ further elucidated the role of GPC1 in the TGF-β signaling pathway, in which they found GPC1 downregulation led to an altered response toward TGF-β1, activin-A, and BMP2 in terms of growth, p21 induction, and Smad2 phosphorylation, resulting in pancreatic cancer cell growth inhibition. Moreover, GPC1 was found to be involved in the activation of the epidermal growth factor receptor (EGFR) signaling pathway in response to EGFR ligands, including heparin-binding epidermal growth factor–like growth factor (HB-EGF) in ESCC cells.⁴¹ Finally, Li et al.⁴² further demonstrated that GPC1 promotes ESCC cell proliferation by regulating the PTEN/Akt/β-catenin pathways. The involvement of GPC1 in the signaling pathways described above is associated with the membrane-bound form of GPC1; whether these activities might be counteracted by the soluble form of GPC1 warrants further investigations.

GPC1 as a Target of Therapy

The anti-GPC1 antibody (clone 1–12) was isolated from chicken and recognized both human and mouse GPC1.⁴¹ It induced 70% tumor growth inhibition in both ESCC xenograft models and patient-derived tumor (PDX) xenograft models in immunodeficient mice. The same research group then isolated another anti-GPC1 mouse MAb, clone 01a033, and developed an antibody–drug conjugate (ADC) by conjugating the MAb with monomethyl auristatin F.³³ The GPC1-ADC showed potent tumor growth inhibition in xenograft models of uterine cervical cancer and pancreatic cancer.^33,43

Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has shown a remarkable response rate exceeding 80% against B-cell malignancies.^44,45 This success has led to many studies evaluating the efficacy of CAR T-cell therapy in solid tumors. Kato et al.⁴⁶ developed GPC1-specific human and murine CAR T-cells generated from the anti-human/mouse GPC1 antibody (clone 1–12) and demonstrated their antitumor effects in xenogeneic and syngeneic solid tumor mouse models without showing any obvious adverse effects. This study shows the potential of GPC1 as an immunotherapeutic target for solid tumors.

GPC2

The GPC2 gene is located on chromosome 7q22, which encodes the GPC2 protein consisting of a 579-amino-acid core protein and five predicted HS chains at S55, S92, S155, S500, and S502 (UniProtKB/Swiss-Prot Q8N158). GPC2 is exclusively expressed in the developing nervous system.⁴⁷ GPC2 is expressed on neuronal growth cones, making it likely that it functions in axon growth and guidance.⁴⁸ After axonal growth has completed, GPC2 is no longer expressed in the nervous system.⁴⁹

GPC2 Expression in Cancer

Orentas et al.⁵⁰ initially reported that GPC2 is highly expressed at the mRNA level in at least eight different pediatric cancers. In particular, GPC2 has been highly expressed in neuroblastoma. We and the Maris group showed that GPC2 protein was highly expressed in several neuroblastoma cell lines by Western blot.^12,13 GPC2 was detected at the cell surface, shown by IHC, in a majority of neuroblastoma tumors, but is not detectable in adult tissues,^12,13 making its expression tumor-specific. Moreover, integration of clinical and genomic information from neuroblastoma patients revealed a correlation between high GPC2 expression and poorer neuroblastoma prognosis, including poorer overall survival.¹²

We also generated a panel of mouse anti-human GPC2 MAbs.⁵¹ Among five hybridoma clones, CT3 demonstrated the best binding affinity to cell surface GPC2 by flow cytometry. As shown in Fig. 1, GPC2 is uniformly overexpressed in neuroblastoma compared with normal peripheral nerve tissue as determined by IHC using the CT3 antibody. Moreover, the CT3 antibody showed the most restricted expression in human normal tissues by IHC among five hybridoma clones. These observations suggest that the CT3 antibody could be useful for neuroblastoma diagnosis.

In clinical settings, neuroblastoma prognosis is partially determined by the status of the oncogene MYCN, which encodes the protein N-Myc, where the presence of MYCN amplification stratifies neuroblastoma cases into a high-risk group.⁵² Neuroblastoma tumors already designated as high-risk had significantly higher GPC2 mRNA expression.¹³ In addition, neuroblastoma cell lines that have high GPC2 protein expression also have high N-Myc protein expression; neuroblastoma cell lines that are GPC2 low-expressing do not express N-Myc, further supporting a possible biological connection between GPC2 and MYCN in neuroblastoma.¹²

Several other pediatric cancers displayed increased GPC2 levels similar to those of neuroblastoma, including medulloblastoma and retinoblastoma.¹³ In particular, group 4 medulloblastoma had GPC2 mRNA expression significantly higher than the other groups.¹³ This group can also have MYCN amplification, where MYCN-amplified cases have a significantly worse outcome.⁵³ In addition, MYCN amplification is also frequently associated with retinoblastoma.⁵⁴

GPC2 Signaling in Cancer

Glypicans are known to regulate Wnt/β-catenin signaling to affect cell growth and proliferation.^6–8 It has been shown that β-catenin can activate the N-Myc promoter during embryonic development.⁵⁵ It is possible that GPC2 functions as a modulator of Wnt signaling, acting to regulate MYCN in neuroblastoma pathogenesis. Indeed, we found that GPC2 could interact with Wnt3a.¹² We demonstrated that genetic silencing of GPC2 inhibited neuroblastoma cell growth by downregulating Wnt/β-catenin signaling to suppress N-Myc expression, indicating that neuroblastoma may be addicted to GPC2 for viability and proliferation. Bosse et al.¹³ found that depleting GPC2 in neuroblastoma cell lines decreased cell proliferation, whereas overexpressing GPC2 increased cell proliferation. In addition, a pathway analysis showed that the Wnt signaling pathway is GPC2-associated.¹³

GPC2 as a Target of Therapy

The tumor-specific expression of GPC2 makes it attractive as a therapeutic target, as off-target effects can be avoided. Tumor escape through downregulation of a tumor antigen is a common problem in immunotherapy.⁵⁶ As neuroblastoma cell proliferation might be dependent on GPC2, a tumor would likely not be able to downregulate its expression. This further supports GPC2 as a promising therapeutic target.

We have developed several antibody-based therapeutics that target GPC2 for the treatment of neuroblastoma. First, anti-GPC2 human single-domain antibodies (e.g., LH7) were isolated via phage display technology. Immunotoxin constructed from LH7 and a 38-kDa fragment of Pseudomonas exotoxin (PE-38) inhibited neuroblastoma tumor growth in mice.¹² Moreover, the GPC2-targeted CAR T-cells we produced with LH7 were able to eliminate tumors in a disseminated neuroblastoma mouse model.¹²

Bosse et al.¹³ developed an ADC that targets GPC2. They screened a human Fab phage display library with GPC2 and found D3-GPC2-Fab binds GPC2 with high affinity. The ADC was constructed using D3-GPC2-IgG1, a human IgG1 converted from D3-GPC2-Fab, and pyrrolobenzodiazepine, a DNA-damaging agent frequently used in cancer treatment. When tested in seven neuroblastoma cell lines, the D3-GPC2-PBD ADC induced cytotoxicity in an antigen-dependent manner. The ADC also showed antitumor efficacy in a neuroblastoma mouse model.

GPC3

The GPC3 gene is on chromosome Xq26 and encodes the GPC3 protein composed of a 580-amino-acid core protein and two predicted HS chains attached at S495 and S509 (UniProtKB/Swiss-Prot P51654). Three variants have been detected that encode alternatively spliced forms,⁵⁷ although the distribution and function of GPC3 isoforms remain largely unknown. GPC3 is expressed in various fetal tissues (liver, lung, kidney, placenta), but is absent or expressed at low levels in most adult tissues.⁵⁸ GPC3 can regulate cell proliferation by modulating Wnt,^8,59,60 YAP,¹⁵ Hedgehog (Hh),^61–63 hepatocyte growth factor (HGF),⁶⁴ and other signaling pathways.

GPC3 Expression in Cancer

In 1997, Hsu et al.⁶⁵ first described the abnormal expression of GPC3 mRNA in 74.8% (143/191) of HCC tissues, compared with only 3.2% (5/154) in non-tumor liver tissues. At the protein level, Capurro et al.⁶⁶ detected GPC3 expression in 72% (21/29) of HCC patients via IHC, whereas no GPC3 expression (0/38) was found in patients with a healthy liver or benign liver diseases. Similar results were confirmed by Baumhoer et al.,⁶⁷ who found GPC3 expression in 63.6% (140/220) of HCC tissues. In addition, GPC3 was detected in HCC tissues but not in benign liver tumors such as dysplastic nodules.^68,69 In patients with hepatitis C virus infection, IHC results showed a 100% positive staining of GPC3 in HCC (n=22) compared with that of dysplastic nodules (0%, n=14).⁷⁰ Many studies have also reported that GPC3 expression is correlated with a poor prognosis in patients with HCC.^71–74 Takai et al.⁷⁵ reported that the expression of GPC3 in HCC was involved in macrophage recruitment, indicating GPC3 may play a role in tumor microenvironment.

We conducted a high-throughput flow cytometry screening to identify a high-affinity anti-GPC3 MAb, YP7, from mouse hybridoma technology.⁷⁶ The YP7 antibody recognizes the C-lobe of GPC3. Our immunohistochemical staining using the YP7 antibody confirmed that the level of GPC3 is highly upregulated in HCC tumor compared with normal liver tissue (Fig. 1).

Furthermore, the overexpression of GPC3 was found in the following adult cancers: lung squamous cell carcinoma (LSCC),⁷⁷ melanoma,⁷⁸ Merkel cell carcinoma,⁷⁹ ovarian clear cell carcinoma (OCCC),⁸⁰ thyroid cancer,⁸¹ urothelial carcinoma,⁸² and salivary gland tumors.⁸³ Moek et al.⁸⁴ performed a functional genomic mRNA profiling to predict GPC3 overexpression at the protein level in 60 cancer types in 18,055 PDX samples and validated the prediction by comparing with IHC staining of a breast cancer tissue microarray. The percentage of samples with predicted GPC3 overexpression was 77% for HCCs (n=364), 45% for LSCCs (n=405), and 19% for head and neck squamous cell carcinomas (n=344). More recently, GPC3 was found to be expressed in glioblastoma and is a driver of glioma tumorigenesis, network hyperexcitability, and synapse formation.⁸⁵

As an oncofetal protein, GPC3 is also highly expressed on a variety of pediatric solid embryonal tumors. Multiple studies have demonstrated that GPC3 expression was increased on most hepatoblastomas.^86–90 GPC3 was found to be overexpressed in germ cell tumors, particularly yolk sac tumors and choriocarcinomas.^90–93 In addition, elevated expression of GPC3 was observed in a significant portion of Wilms tumors compared with adult kidney tumors and normal kidney tissue.^86,94,95 Somatic mutations in GPC3 have been identified in some cases of Wilms tumors.⁹⁶ Moreover, 25% to 35% of rhabdomyosarcoma (RMS) cases showed GPC3-positive staining from three different studies, whereas other pediatric sarcomas often do not express GPC3.^89,90,97

Nevertheless, GPC3 was found to act as a potential tumor suppressor in some cancers. In a study done on the loss-of-function mutation of GPC3 in embryonic development, researchers observed an overgrowth of organs in fetal and postnatal development, known as Simpson–Golabi–Behmel overgrowth syndrome (SGBS).^98,99 The reduced or even silenced expression of GPC3 was found in other cancer types including renal cell carcinoma, breast cancer, ovarian cancer, and mesothelioma.^100–103 In breast cancer, ovarian cancer, and mesothelioma, the loss of GPC3 expression was partially due to hypermethylation of its promoter region, and its expression was restored by treating cells with a demethylating agent.^100,102,103 Ectopic expression of GPC3 also induced the apoptosis of lung cancer cells and inhibited tumor growth in mice.¹⁰⁴ Overall, these findings suggest that GPC3 plays different roles in a tissue-dependent manner in various cell lineages.

As GPC3 is highly expressed in HCCs, it is important to understand the regulatory mechanisms of GPC3 expression during HCC tumorigenesis. Trinh et al.¹⁰⁵ showed that epigenetic alternations including both DNA methylation and histone modifications were critical for the transcriptional regulation of GPC3 in HCC. Li et al.¹⁰⁶ demonstrated that GPC3 is a direct transcriptional target of c-Myc and that c-Myc overexpression increased GPC3 levels. Conversely, increased levels of GPC3 also elevated c-Myc expression, thus forming a positive feedback signaling loop. In addition, Maurel et al. screened a library of 876 microRNAs (miRNAs) and identified five miRNAs that regulate GPC3 expression: miR-96 and miR-1271 showed negative regulation, whereas miR-129-1-3p, miR-1291, and miR-1303 played inducing roles.^107,108

GPC3 Signaling in Cancer

GPC3 acts as an essential coreceptor and triggers the activation of signaling, including Wnt, Hh, FGF, and HGF pathways in cancer.

Wnt Signaling

Activation of the Wnt signaling pathway is one of the most frequent molecular events associated with the progression of HCC. Approximately 95% of HCCs exhibit Wnt/β-catenin deregulation.¹⁰⁹ Both the core protein and the HS chains may be involved in GPC3-modulated Wnt signaling. It has been shown that GPC3 HS chains may not be required for Wnt binding,⁵⁹ but are essential for its interaction with frizzled (FZD).⁶ We identified a Wnt-recognizing motif on the HS chains of GPC3 using the HS20 antibody that binds the HS with high affinity and blocks GPC3 and Wnt3a interaction, and found that 2-O and 6-O sulfations were essential for Wnt binding while 3-O sulfation could enhance Wnt binding.^7,110 Evidence of the Wnt binding domain on the GPC3 core protein has been indicated using the HN3 antibody.⁶⁰ More recently, we identified F41, a key residue within GPC3’s cysteine-rich hydrophobic groove located in the N-lobe of GPC3, which is critical for the GPC3/Wnt interaction.⁸ We hypothesize that GPC3 serves as a Wnt coreceptor or receiver to attract Wnt to the cell surface and acts as a transmitter through its HS chains to stabilize the Wnt/GPC3/FZD tripartite complex once it is formed, thereby triggering downstream signaling (Fig. 2). Therefore, we believe GPC3 functions as both a receiver and a transmitter on the cell surface for Wnt signals.

Figure 2.

The model for the role of GPC3 in modulating Wnt signaling in HCC. GPC3 functions as a Wnt receiver to attract Wnt to the cell surface and acts as a transmitter through its HS chains to stabilize the Wnt/GPC3/FZD tripartite complex once it is formed, which triggers downstream β-catenin signaling. RSPO binds to the HS chains of glypican (e.g. GPC3) and the E3 ligases RNF43/ZNRF3 to block the degradation of FZD and LRP, thereby potentiating Wnt signaling. Abbreviations: FZD, frizzled; HCC, hepatocellular carcinoma; HS, heparan sulfate; LRP, lipoprotein receptor related protein; RSPO, R-spondin; TCF, T cell factor.

Cell Growth Factors

GPC3 has been demonstrated to interact with growth factors through its HS chains and ultimately stimulate cell growth.¹¹¹ In HCC cells, GPC3 binds to FGF-2 through the HS chains.¹¹² We demonstrated that GPC3 functions in HCC cell migration and motility through HS chain–mediated cooperation with the HGF/c-Met pathway⁷; antibody targeting the GPC3 core protein had no effect on HCC motility.⁶⁴ Sun et al.¹¹³ discovered that GPC3 suppression in HCC cells led to enhanced TGF-β2 expression and thereby inhibited cell proliferation, arrested cell cycle progression, and induced replicative senescence. Midorikawa et al.¹¹¹ showed that GPC3 overexpression inhibited BMP7 signaling and modulated BMP7 activity through the SMAD pathway and thus promoted cell proliferation.

Hh Signaling

GPC3 is a potent inhibitor of Hh signaling. Loss-of-function mutations of GPC3 cause SGBS, partially due to hyperactivation of the Hh pathway.^114,115 Capurro et al.⁶² showed that GPC3 bound to sonic hedgehog (Shh) and Indian hedgehog (Ihh) with high affinity in an HS-independent manner, resulting in a strong competition with Patched (Ptc) for Hh binding. In a later study, they demonstrated that cleavage by convertases is also crucial for GPC3 inhibition of Hh signaling.⁶¹ The low-density lipoprotein receptor–related protein 1 (LRP1) was also shown to mediate endocytosis of the GPC3/Hh complex.¹¹⁶

YAP Signaling

HCC tissues are reported to show higher YAP activation, indicating a positive correlation between HCC progression and YAP activity.¹¹⁷ We found that knockdown of GPC3 induced apoptosis of HCC cells and reintroduction of constitutively active mutant YAP (S127A) was able to rescue HCC cells from apoptosis.¹⁵ We also demonstrated that the HN3 single-domain antibody inhibited HCC cell growth by inactivating YAP signaling.^15,60

GPC3 displays a tissue-specific pattern of expression during tumor progression. It is overexpressed in some cancers (e.g., HCC) but acts as a tumor suppressor in other cancers (e.g., breast cancer). It is speculated that the tissue-specific differences are related to the ability of GPC3 to regulate different growth factors and signaling pathways.^115,118 For example, the overexpression of GPC3 in HCC will promote tumor growth by stimulating the Wnt signaling pathway, which plays a vital role in HCC progression. In contrast, GPC3 exhibits an opposite role acting as an inhibitor in Hh signaling. The loss of GPC3 may contribute to cancer progression by inducing the activation of Hh signaling. It has been reported that the Hh signaling pathway is hyperactivated in breast and ovarian cancers.^119,120

GPC3 as a Target of Therapy

Given the tumor specificity, GPC3 is an ideal target for cancer immunotherapy. Various therapeutic strategies have been developed to target GPC3, which are described in more detail below.

Vaccines

Peptide vaccines of GPC3 have been extensively investigated in preclinical and clinical studies. Initially, several reports showed that peptide-specific cytotoxic T-lymphocytes (CTLs) were induced in mice immunized with HLA-A24- and HLA-A2-restricited GPC3-derived peptides, which exerted antitumor effects without causing an autoimmune response.^121,122 The first-in-human study of GPC3 peptide vaccine was conducted in Japan, which confirmed the safety of vaccines and demonstrated the GPC3 peptide–specific immune responses.^123,124 In a later phase II clinical trial, Sawada et al.¹²⁵ demonstrated that postsurgical administration of GPC3 peptide vaccine extended the recurrence-free survival period, and overall survival was prolonged in cases where CTL induction was observed. The same group developed GPC3-derived long peptides (GPC3-LPs) carrying human leukocyte antigen class II–restricted T-helper cell epitopes, which induced peptide-specific CD4⁺ T-cell responses from most healthy donors.¹²⁶ In addition, clinical trials with a GPC3-derived peptide vaccine were conducted in patients with OCCC and hepatoblastoma.^127,128 In both cancer types, vaccines induced GPC3-specific CTL responses in patients and extended recurrence-free survival.

Monoclonal Antibodies

Several therapeutic anti-GPC3 antibodies have been developed in recent years. GC33, a mouse MAb against GPC3, has been shown to induce antibody-dependent cell-mediated cytotoxicity against GPC3-positive HCC cell lines and inhibit tumor growth in HCC xenograft mouse models.^129,130 In a first-in-human phase I trial for patients with advanced HCC, GC33 was well tolerated and antitumor effects were observed in some patients with high GPC3 expression.¹³¹ However, it did not show significant clinical benefit in a randomized phase II clinical trial,¹³² suggesting that antibodies alone may have limited therapeutic effects in HCC patients.

Our laboratory has isolated several anti-GPC3 antibodies for cancer therapy, including YP7, HN3 and HS20, which have been widely used preclinically. Although YP7 recognizes the C-lobe of GPC3 protein, it has a distinct binding epitope from GC33 based on our recent study.¹³³ We also humanized the YP7 antibody (hYP7) using antibody engineering¹³⁴ and showed that both YP7 and hYP7 antibodies had considerable antitumor activity in HCC xenograft mouse models.^76,134 HN3, a human single-domain antibody that targets the N-lobe of GPC3, induced cell cycle arrest in HCC cells via inhibition of Wnt and YAP signaling.^15,60 HS20 is a human MAb isolated by phage display technology, which recognizes HS but not chondroitin sulfate.^7,110,135 The binding of the HS20 antibody to HS requires sulfation at both C2 position and C6 position. HS20 could disrupt the GPC3/Wnt interaction, thereby inhibiting Wnt/β-catenin signaling and suppressing the growth of HCC xenografts in mice.^7,110

Immunotoxins

To improve the potency of MAb, we have made numerous efforts to develop novel GPC3-targeted immunotoxins. Our first study showed that HN3-PE38 immunotoxin regressed HCC xenograft tumors in mice at 0.6 mg/kg dosage.⁶⁰ To reduce the side effects and immunogenicity, we constructed two deimmunized immunotoxins, HN3-mPE24 and HN3-T20, that contain only domain III of the PE38 toxin. In one approach, we removed B-cell epitopes on domain III and constructed HN3-mPE24 which was well tolerated in mice even at 5 mg/kg dosage and induced significant tumor regression in mice bearing HCC xenograft.¹³⁶ In another approach, we engineered domain III by removing T-cell epitopes, creating the immunotoxin HN3-T20, which showed a greater increase in overall survival than HN3-mPE24.¹³⁷ To enhance serum retention, we then added a streptococcal albumin-binding domain (ABD) into HN3-T20 and found that HN3-ABD-T20 had a 45-fold higher serum half-life than HN3-T20. Although further clinical investigation is required, the current data identify GPC3-targeted immunotoxins as promising candidates for HCC therapy.

CARs

The GPC3-targeted CARs based on the GC33 single-chain variable fragment (scFv) were initially reported by Gao et al.¹³⁸ They demonstrated that GPC3-targeted CAR T-cells could inhibit the growth of orthotopic HCC xenografts in mice. Li et al.¹³⁹ compared the contribution of CD28 and 4-1BB by evaluating the antitumor activities of T-cells expressing GC33-based CAR targeting GPC3. T-cell signaling through CD28 had higher cytotoxicity than the signaling through 4-1BB in vitro; however, 4-1BB induced the superior expansion of CAR T-cells and demonstrated potent antitumor activity in vivo. Jiang et al.¹⁴⁰ found that GPC3-targeted CAR T-cells effectively eliminated tumors in PDX model of HCC. In addition, Wang et al.¹⁴¹ used the PiggyBac transposon system to deliver the GPC3-targeted CAR into T-cells, which reduced tumor burden in an HCC xenograft mouse model. Moreover, Chen et al. found that dual-targeted CAR T-cells coexpressing GPC3 and asialoglycoprotein receptor 1, a liver tissue–specific protein, reduced the risk of on-target/off-tumor toxicity in vivo. Furthermore, the same research group demonstrated that disruption of PD-1 enhanced the antitumor activity of GPC3-targeted CAR T-cells against HCC in mice and improved the persistence of infiltration of CAR T-cells.^142,143 Based on these findings, Shi et al.¹⁴⁴ have begun a phase I trial of HCC patients in China and demonstrated that GPC3-targeted CAR T-cell therapy was feasible for the first time. Early signs of antitumor activity were also observed in patients with advanced HCC.

We have developed CARs based on the hYP7 and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively.¹³³ Injection of CAR (hYP7) T-cells eliminated tumors in mice bearing orthotopic HCC xenografts, whereas CAR (HN3) T-cells did not reduce tumor burden. Mice injected with CAR (hYP7) T-cells had persistent expansion of T-cells that were found in spleen for up to 7 weeks after infusion and in subsets of polyfunctional CAR T-cells via antigen-induced selection. In the same study, we analyzed the integration sites of CAR (hYP7) in mice to investigate candidate factors contributing to CAR T-cell expansion and persistence. Many shared integrated genes were observed in different tissues on the same mouse, likely due to the expansion of selected clones. Interestingly, several integration events (e.g., NUPL1, DENND1B) became highly abundant in multiple mice at weeks 5 and 7 after infusion, indicating certain CAR-integrated sites or genes might play roles in CAR (hYP7) T-cell activation, survival, and expansion in vivo. The clinical development of CAR T-cell therapy against GPC3 based on our research is underway in China (NCT04121273) and the United States (NIH Clinical Center).

ADCs

In addition to immunotoxins and CARs, we constructed the GPC3-specific ADCs using the hYP7 antibody.¹⁴⁵ HCC is known to have resistance to cancer chemotherapeutics.^146,147 To determine the payload for ADCs against liver cancer, we and collaborators screened three large drug libraries (>9000 compounds) against HCC cell lines and found that the most potent drugs are DNA-damaging agents. We chose duocarmycin SA and pyrrolobenzodiazepine dimer as the payloads to construct two GPC3-specific ADCs: hYP7-DC and hYP7-PC. Encouragingly, single treatment of hYP7-PC induced tumor regression in multiple HCC xenograft mouse models. Lipovsek et al.¹⁴⁸ conjugated an Adnectin that binds GPC3 to a cytotoxic derivative of tubulysin and proved its fast clearance from normal tissues and efficacy against GPC3-positive xenograft mouse models.

Other Approaches

MAbs can also be conjugated to the photosensitizing phthalocyanine dye IR700 to target cancer cells through exposure to near-infrared light. The conjugation of IR700 with our YP7 and HN3 antibodies, IR700-YP7 and IR700-HN3, showed an antitumor effect in GPC3-positive tumor-bearing mice.¹⁴⁹ Furthermore, the combination of IR700-YP7 and nab-paclitaxel drastically increased nab-paclitaxel delivery and enhanced the therapeutic efficacy.¹⁵⁰

ERY974, an anti-GPC3/CD3 bispecific T-cell-redirecting antibody regressed GPC3-positive solid tumors in mice.¹⁵¹ Intriguingly, ERY974 was effective even against tumors with non-immunogenic features by causing inflammation in the local tumor microenvironment. Bi et al.¹⁵² also reported a bispecific T-cell engager (BiTE) targeting GPC3, and CD3 could efficiently mediate the T-cell killing of GPC3-positive HCC cells in vitro and in vivo.

A dominant T-cell receptor (TCR), P1-1, targeting an abundantly displayed HLA-A2-specific GPC3 peptide was cloned from CTLs that were induced by dendritic cells overexpressing HLA-A2 and GPC3.¹⁵³ CTLs overexpressing P1-1 inhibited GPC3-positive xenograft tumor growth at early stages, but its effect decreased in vivo in later phases. In addition, GPC3-specific CAR natural killer cells inhibited 90% of GPC3-expressing HCC xenograft growth.¹⁵⁴

GPC4

GPC4 is composed of a 556-amino-acid core protein with the attachment of three HS chains at S494, S498, and S500 (UniProtKB/Swiss-Prot O75487). Interestingly, GPC4 is adjacent to the 3′ end of GPC3 on chromosome Xq26.¹⁵⁵ GPC4 shows a predominant expression in developing brain and kidney.¹⁵⁶

GPC4 Expression in Cancer

The increased expression of GPC4 has been found in pancreatic cancer. It is also involved in 5-fluorouracil resistance and pancreatic cancer stemness,¹⁵⁷ suggesting that GPC4 may serve as a strategy for pancreatic cancer therapy. Fang et al.¹⁵⁸ found that GPC4 protein level was significantly upregulated in colorectal tumor tissues compared with matched adjacent normal mucosa. In addition, recent studies demonstrated that GPC4 gene polymorphism was associated with susceptibilities to Epstein-Barr virus (EBV)-associated gastric carcinoma¹⁵⁹ and EBV-positive nasopharyngeal carcinoma in Northern China.¹⁶⁰ Moreover, Varma et al.¹⁶¹ showed that GPC4 mRNA level was downregulated in oxaliplatin-resistant ovarian carcinoma cell line A2780/C10. Recently, Munir et al.¹⁶² demonstrated that GPC4 expression was downregulated in metastatic breast tumors compared with non-metastatic breast tumors. Their results also indicate that GPC4 acts as a tumor suppressor in breast cancer by decreasing migration and proliferation.

Although mutations in GPC3 have been causatively and genetically linked to the SGBS, it has been speculated that GPC4, which is close to GPC3 on the X chromosome, might also be involved in the SGBS or other X-linked overgrowth or undergrowth syndromes. In one study, analysis of DNA samples from eight SGBS patients identified one individual with a deletion of both 3′ end of GPC3 and the entire GPC4 gene.¹⁵⁵ In another report, a duplication GPC4 was identified in SGBS patients with no mutation found in GPC3,¹⁶³ indicating GPC4 might independently play a role in growth control.

GPC4 Signaling in Cancer

Like other glypicans (e.g., GPC2 and GPC3), GPC4 could also be a regulator of Wnt signaling. Sakane et al.¹⁶⁴ demonstrated that GPC4 is colocalized with different Wnts to lipid raft and non-lipid raft microdomains and thereby regulates distinct Wnt signaling pathways. Recently, Fang et al.¹⁵⁸ reported that GPC4 interacted with CD36, a membrane glycoprotein involved in diverse cellular functions,¹⁶⁵ in colorectal cancer. CD36–GPC4 interaction promoted the proteasome-dependent ubiquitination of GPC4, followed by inhibition of Wnt/β-catenin signaling.

R-spondins (RSPOs) are secreted proteins that act as potent amplifiers of the Wnt/β-catenin signaling. Lebensohn and Rohatgi¹⁶⁶ found that HSPGs such as GPC4 and GPC6 are required for RSPO-enhanced Wnt signaling in cells lacking leucine-rich repeat-containing G-protein-coupled receptors (LGRs) (LGR4/5/6 knockout). Knockout of exostosin-like 3 (encoded by the EXTL3 gene), a glycosyltransferase required for the biosynthesis of HS chains on HSPGs, reduced ~80% of LGR-independent Wnt signaling. The TSP/BR domain of RSPO3 is proposed as a binding site for HSPGs because the deletion of this domain significantly reduced Wnt signaling activity. A more recent study showed that mutations of key residues in the TSP/BR domain of RSPO3 for HSPG binding also largely reduced Wnt signaling. Furthermore, the loss of Wnt activity in the presence and absence of LGRs by deletion or mutations of the TSP/BR domain of RSPO3 was rescued by replacing the entire TSP/BR domain with the HS20 scFv antibody^64,135 that recognizes HS chains of GPC3 and GPC4.¹⁶⁷ The fact that HSPGs can potentiate Wnt signaling in the absence of LGR (LGR-independent pathway) further supports the important role of GPC4 and other HSPGs as potent Wnt modulators (Fig. 2). Further studies will be needed to analyze precise and potentially differential roles of glypicans in cells containing and lacking LGRs.

GPC5

The GPC5 gene is located on chromosome 13q32, and encodes the GPC5 protein, which consists of a 572-amino-acid core protein and five predicted HS chains attached at S441, S486, S495, S507 and S509 (UniProtKB/Swiss-Prot P78333). GPC5 is expressed in kidney, limb, and brain during development; its expression in brain persists into adulthood.^168,169

GPC5 Expression in Cancer

It has been previously reported that the GPC5 gene was amplified and its mRNA expression was upregulated in RMS.¹⁷⁰ Nishimura et al.¹⁷¹ further confirmed that GPC5 was located in the frequently amplified region in RMS cases, suggesting aberrant GPC5 activity might play a role in RMS oncogenesis.

A genome-wide association study identified a single-nucleotide polymorphism (SNP) in the GPC5 gene associated with susceptibility to lung cancer in never-smokers.¹⁷² Downregulation of GPC5 may contribute to the development of lung cancer in never-smokers. Additional report also demonstrated that GPC5 expression was decreased in non–small cell lung cancer (NSCLC), and overexpression of GPC5 suppressed the migration, invasion, and proliferation of NSCLC cells.¹⁷³ Yuan et al.¹⁷⁴ further confirmed the low expression of GPC5 was significantly associated with poor outcome in NSCLC. They also found that the GPC5 promoter was hypermethylated in lung cancer tissues compared with normal lung tissues, which explains the loss of GPC5 expression.

However, another study found that GPC5 was upregulated in NSCLC and was associated with poor overall survival.¹⁷⁵ Thus, the role of GPC5 in tumorigenesis of lung cancer needs to be further investigated.

In addition, a growing number of studies have demonstrated that GPC5 expression is downregulated in multiple cancers beyond lung cancer. For example, reduced GPC5 expression was found in breast cancer and was associated with poor survival.¹⁷⁶ Zhang et al.¹⁷⁷ recently reported that the level of GPC5 in prostate cancer tissues was significantly lower than that in normal controls, especially in high-risk prostate cancer. The reduced expression of GPC5 has been observed in pancreatic cancer,¹⁷⁸ HCC,¹⁷⁹ and glioma,¹⁸⁰ indicating the tumor suppressive role of GPC5.

GPC5 Signaling in Cancer

GPC5 is reported to be involved in regulating various pathways, including Wnt, Hh, and FGF signaling. GPC5 has been shown to promote RMS proliferation through increasing the intracellular signaling of FGF-2 and HGF.¹⁷⁰ Li et al.¹⁸¹ showed that GPC5 stimulates RMS cell proliferation by promoting Hh signaling. In particular, the tumor suppressive role of GPC5 is extensively associated with its inhibitory effect on Wnt/β-catenin signaling.^174,182,183 Yuan et al.¹⁷⁴ demonstrated that GPC5 could block the activation of Wnt/β-catenin signaling by binding to Wnt3a at the cell surface, which mediates its function as a tumor suppressor. In addition, Wang et al.¹⁸² reported that GPC5 suppressed lung cancer cell metastasis by competitively binding to Wnt3a. Moreover, GPC5 is shown to inhibit Wnt/β-catenin signaling in glioma, prostate, and pancreatic cancer.^178,180,184

Table 1.

Expression of Glypicans in Cancers.

Glypican	Cancer Type	Sample Type	Aberrant Expression		Sites and Localization	Ref.
Glypican	Cancer Type	Sample Type	mRNA	Protein	Sites and Localization	Ref.
GPC1	Pancreatic cancer	Cells, tissues	Increase	Increase	Cancer cell, fibroblast	Kleeff et al., 1998²³
		Tissues	NA	Increase 56% (35/62)	Cancer cell, fibroblast	Duan et al., 2013²⁴
		Tissues	NA	Increase 60% (111/186)	Cell membrane and cytoplasm	Lu et al., 2017²⁵
	Breast cancer	Cells, tissues	Increase	Increase	Cell membrane	Matsuda et al., 2001²⁷
	Glioma	Cells	Increase	NA	NA	Su et al., 2006²⁸
	Glioblastoma	Tissue	NA	Increase 51% (27/53)	NA	Saito et al., 2017²⁹
	Prostate cancer	Cells, tissues	Increase	Increase	Tumor stroma	Suhovskih et al., 2013³⁰
	ESCC	Cells, tissues	NA	Increase 99% (172/175)	Cell membrane	Hara et al., 2016³²
	Uterine cervical cancer	Cells, tissues	NA	Increase	Cell membrane	Matsuzaki et al., 2018³³
GPC2	Pediatric cancers	Tissues	Increase	NA	Cell membrane	Orentas et al., 2012⁵⁰
	Neuroblastoma	Cells, tissues	NA	Increase 52% (13/25)	Cell membrane	Li et al., 2017¹²
	Neuroblastoma	Cells, tissues	Increase	Increase 93% (91/98)	Cell membrane	Bosse et al., 2017¹³
	Medulloblastoma	Cells, tissues	Increase	Increase 90% (57/63)	Cell membrane	Bosse et al., 2017¹³
GPC3	HCC	Tissues	Increase 75% (143/191)	NA	NA	Hsu et al., 1997⁶⁵
		Tissues	NA	Increase 72% (21/29)	Cell membrane	Capurro et al., 2003⁶⁶
		Tissues	NA	Increase 64% (140/220)	NA	Baumhoer et al., 2008⁶⁷
		Tissues	Increase	Increase 76% (36/47)	Cell membrane and cytoplasm	Libbrecht et al., 2006⁶⁹
		Tissues	NA	Increase 60% (24/40)	Cell membrane and cytoplasm	Li et al., 2020¹³³
	LSCC	Tissues	Increase	Increase 55% (17/31)	Cell membrane and cytoplasm	Aviel-Ronen et al., 2008⁷⁷
	Melanoma	Cells, tissues	Increase	Increase	Cytoplasm	Nakatsura et al., 2004⁷⁸
	Merkel cell carcinoma	Tissues	NA	Increase 55% (17/31)	Cell membrane and cytoplasm	He et al., 2009⁷⁹
	OCCC	Tissues	NA	Increase 44% (41/94)	Cell membrane and cytoplasm	Maeda et al., 2009⁸⁰
	Thyroid cancer	Tissues	Increase	Increase	Cell membrane and cytoplasm	Yamanaka et al., 2007⁸¹
	Urothelial carcinoma	Tissues	NA	Increase 35% (38/108)	NA	Aydin et al., 2015⁸²
GPC3	Salivary gland tumors	Tissues	NA	Increase	Cell membrane and cytoplasm	Andisheh-Tadbir et al., 2019⁸³
	Glioblastoma	Tissues	NA	Increase	NA	Yu et al., 2020⁸⁵
	Hepatoblastoma	Tissues	NA	Increase100% (9/9)	NA	Chan et al., 2013⁸⁹
		Tissues	NA	Increase60% (6/9)	NA	Kinoshita et al., 2015⁹⁰
		Tissues	Increase	NA	NA	Toretsky et al., 2001⁸⁶
		Tissues	NA	Increase100% (65/65)	Cytoplasm	Zynger et al., 2008⁸⁸
	YST	Tissues	NA	Increase91% (10/11)	NA	Kinoshita et al., 2015⁹⁰
		Tissues	NA	Increase97% (31/32)	Cell membrane and cytoplasm	Esheba et al., 2008⁹¹
		Tissues	NA	Increase100% (24/24)	Cell membrane and cytoplasm	Zynger et al., 2006⁹²
		Tissues	NA	Increase100% (33/33)	Cell membrane and cytoplasm	Zynger et al., 2008⁹³
	choriocarcinoma	Tissues	NA	Increase100% (7/7)	Cell membrane and cytoplasm	Zynger et al., 2006⁹²
	Wilms tumor	Tissues	Increase	NA	NA	Toretsky et al., 2001⁸⁶
		Tissues	Increase	NA	NA	Saikali et al., 2000⁹⁴
		Tissues	Increase	Increase77% (23/30)	Cell membrane and cytoplasm	Tretiakova et al., 2015⁹⁵
	RMS	Tissues	NA	Increase23% (19/82)	NA	Chan et al., 2013⁸⁹
		Tissues	NA	Increase25% (14/56)	NA	Kinoshita et al., 2015⁹⁰
		Tissues	Increase	Increase35% (74/213)	Cell membrane and cytoplasm	Thway et al., 2011⁹⁷
	Mesothelioma	Cells, tissues	Decrease	NA	NA	Murthy et al., 2000¹⁰⁰
	Renal CCC	Cells, tissues	Decrease	NA	NA	Valsechi et al., 2014¹⁰¹
GPC4	Pancreatic cancer	Tissues	NA	Increase	NA	Cao et al., 2018¹⁵⁷
	Colorectal cancer	Tissues	NA	Increase	NA	Fang et al., 2019¹⁵⁸
	Drug-resistant ovarian cancer	Cells	Decrease	NA	NA	Varma et al., 2005¹⁶¹
	Breast cancer	Cells	Decrease	Decrease	NA	Munir et al., 2020¹⁶²
GPC5	RMS	Tissues	Increase	NA	NA	Williamson et al., 2007¹⁷⁰
	NSCLC	Cells, tissues	Increase	Increase46% (58/127)	Cell membrane	Li et al., 2013¹⁷⁵
		Cells, tissues	Decrease	Decrease	NA	Yang et al., 2013¹⁷³
		Cells, tissues	Decrease	Decrease	NA	Yuan et al., 2016¹⁷⁴
GPC5	Breast cancer	Tissues	Decrease	NA	NA	Zhang et al., 2011¹⁷⁶
	Prostate cancer	Tissues	NA	Decrease	NA	Zhang et al., 2016¹⁷⁷
	Pancreatic cancer	Cells	NA	Decrease	NA	Yuan et al., 2018¹⁷⁸
	Glioma	Cells	NA	Decrease	NA	Hong et al., 2019¹⁸⁰
GPC6	Gastric cancer	Tissues	Increase	NA	NA	Dinccelik-Aslan et al., 2015¹⁸⁶
	Ovarian cancer	Cells, tissues	Increase	Increase	NA	Karapetsas et al., 2015¹⁸⁷
	Melanoma	Cells	Increase	NA	NA	Li et al., 2019¹⁸⁹
	Drug-resistant ovarian cancer	Cells	Decrease	NA	NA	Januchowski et al., 2014¹⁹⁰
	Retinoblastoma	Tissues	Decrease	NA	NA	Lau et al., 2010¹⁹¹

Abbreviations: GPC, glypican; CCC, clear cell carcinoma; ESCC, esophageal squamous cell carcinoma; HCC, hepatocellular carcinoma; LSCC, lung squamous cell carcinoma; NA, not available; NSCLC, non-small cell lung cancer; OCCC, ovarian clear cell carcinoma; RMS, rhabdomyosarcoma; YST, yolk sac tumor.

GPC6

The GPC6 gene is colocalized with GPC5 on chromosome 13q32. It encodes the GPC6 protein composed of a 555-amino-acid core protein and an unknown number of HS chains (UniProtKB/Swiss-Prot Q9Y625). GPC6 is most homologous to GPC4 (63%). Like other glypicans that are expressed during embryonic development, GPC6 shows a high expression in human fetal kidney.¹⁸⁵ It is also widely expressed in adult tissues with the most abundance in ovary.¹⁸⁵

GPC6 Expression in Cancer

The role of GPC6 has been implicated in cancer. The expression of GPC6 mRNA was found to be significantly higher in gastric cancer tissues compared with that in normal tissues; however, there was no statistically significant association between GPC6 expression and pathological parameters studied.¹⁸⁶ Karapetsas et al.¹⁸⁷ reported that GPC6 mRNA was overexpressed in early-stage ovarian cancer and its expression level correlated with CD8⁺ T-cell infiltration. They also found that the mRNA level of GPC6 was associated with overall survival of patients with early-stage ovarian cancer. A recent study reported that hypoxia-induced HIF1α in melanocytes directly regulated the expression of GPC6 and that increased expression of GPC6 was positively associated with poor survival in melanoma.¹⁸⁸ Li et al.¹⁸⁹ further identified GPC6 as one of the top genes whose expression levels distinguished primary melanoma from metastatic melanoma, indicating GPC6 could be a biomarker for melanoma progression. In addition, GPC6 expression has been reported to be reduced in certain cancer types. Januchowski et al.¹⁹⁰ showed that GPC6 was strongly downregulated in chemoresistant variants of the A2780 ovarian cancer cell line. Lau et al.¹⁹¹ found that a reduction of GPC6 mRNA level in retinoblastoma was associated with the non-random allelic loss at chromosome 13q31, which could contribute to retinoblastoma development.

Alternations of GPC6 DNA in cancer have also been identified in recent years. Kumar et al.¹⁹² identified that GPC6 was recurrently mutated across individual prostate tumors from different patients. In another large-scale genome-wide association study, Amankwah et al.¹⁹³ identified an SNP (rs17702471) in GPC6/GPC5 that was associated with an increased risk of invasive epithelial ovarian cancer.

GPC6 Signaling in Cancer

Similar to the interaction between GPC4 and RSPO, GPC6 is also required for RSPO-enhanced Wnt signaling at low levels of Wnt¹⁹⁴ or in the absence of LGRs.¹⁶⁶ In addition, Yiu et al.¹⁹⁵ found that nuclear factor of activated T-cells (NFAT) transcriptionally regulates GPC6 induction in breast cancer, which leads to increased breast cancer cell invasion and migration by upregulating non-canonical Wnt5a signaling.

In conclusion, glypicans are very important in the modulation of growth factors and signaling pathways, such as Wnt signaling. They are expressed abnormally in various cancerous tissues and involved in tumorigenesis. In this review, we summarized the abnormal expression of glypicans in cancers and discussed their potentials as diagnostic tools and therapeutic targets in cancers. Glypicans have also emerged as novel tumor antigens. The therapeutic options involving glypican-targeted therapies are promising. There are currently multiple ongoing preclinical and clinical studies of glypicans-targeted therapies, which will validate and corroborate the role of glypicans as emerging cancer therapeutic targets.

Footnotes

Acknowledgements

We thank the National Institutes of Health Fellows Editorial Board for editorial assistance. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. The YP7, CT3, and HM2 antibodies are available for licensing, in a wide range of fields of use, from the National Cancer Institute. If you are interested in obtaining a license, please contact the principal investigator at homi@mail.nih.gov.

Competing Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author Contributions

NL and MS reviewed the literature and wrote the manuscript; MH revised and approved the final manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Intramural Research Program of National Institutes of Health, National Cancer Institute (Z01 BC010891 and ZIA BC010891 to M.H).

References

Ferlay

Colombet

Soerjomataram

Mathers

Parkin

Pineros

Znaor

Bray

. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019 Apr 15;144(8):1941–53.

Knelson

Nee

Blobe

. Heparan sulfate signaling in cancer. Trends Biochem Sci. 2014 Jun;39(6):277–88.

Nagarajan

Malvi

Wajapeyee

. Heparan sulfate and Heparan sulfate proteoglycans in cancer initiation and progression. Front Endocrinol (Lausanne). 2018;9:483.

Bernfield

Gotte

Park

Reizes

Fitzgerald

Lincecum

Zako

. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem. 1999;68:729–77.

Sarrazin

Lamanna

Esko

. Heparan sulfate proteoglycans. Cold Spring Harb Perspect Biol. 2011 Jul 1;3(7):a004952.

Capurro

Martin

Shi

Filmus

. Glypican-3 binds to Frizzled and plays a direct role in the stimulation of canonical Wnt signaling. J Cell Sci. 2014 Apr 1;127(Pt 7):1565–75.

Gao

Kim

Feng

Phung

Xavier

Rubin

. Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy. Hepatology. 2014 Aug;60(2):576–87.

Wei

Liu

Bai

Baxa

Wang

Chen

Feng

Lee

Gao

. A Frizzled-like cysteine-rich domain in Glypican-3 mediates Wnt binding and regulates hepatocellular carcinoma tumor growth in mice. Hepatology. 2019 Oct;70(4):1231–45.

Fico

Maina

Dono

. Fine-tuning of cell signaling by glypicans. Cell Mol Life Sci. 2011 Mar;68(6):923–9.

10.

Filmus

Capurro

Rast

. Glypicans. Genome Biol. 2008;9(5):224.

11.

Gao

Zhang

. Glypicans as cancer therapeutic targets. Trends Cancer. 2018 Nov;4(11):741–54.

12.

Hewitt

Dimitrov

. Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma. Proc Natl Acad Sci U S A. 2017 Aug 8;114(32):E6623–31.

13.

Bosse

Raman

Zhu

Lane

Martinez

Heitzeneder

Rathi

Kendsersky

Randall

Donovan

Morrissy

Sussman

Zhelev

Feng

Wang

Hwang

Lopez

Harenza

Wei

Pawel

Bhatti

Santi

Ganguly

Khan

Marra

Taylor

Dimitrov

Mackall

Maris

. Identification of GPC2 as an oncoprotein and candidate immunotherapeutic target in high-risk neuroblastoma. Cancer Cell. 2017 Sep 11;32(3):295–309.e12.

14.

Kim

. Glypican-3: a new target for cancer immunotherapy. Eur J Cancer. 2011 Feb;47(3):333–8.

15.

Feng

Gao

Wang

Chen

Man

Figg

Wang

Dimitrov

. Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma. Proc Natl Acad Sci U S A. 2013 Mar 19;110(12):E1083–91.

16.

Truong

Justiniano

Nocon

Soon

Wissmueller

Campbell

Walsh

. Glypican-1 as a biomarker for prostate cancer: isolation and characterization. J Cancer. 2016;7(8):1002–9.

17.

Awad

Logan

Mani

. GPC1 (glypican 1). Atlas Genet Cytogenet Oncol Haematol. 2014;18(7):461–4.

18.

Qiao

Meyer

Mundhenke

Drew

Friedl

. Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 signaling in brain endothelial cells. Specific role for glypican-1 in glioma angiogenesis. J Biol Chem. 2003 May 2;278(18):16045–53.

19.

Chamorro-Jorganes

Araldi

Rotllan

Cirera-Salinas

Suarez

. Autoregulation of glypican-1 by intronic microRNA-149 fine tunes the angiogenic response to FGF2 in human endothelial cells. J Cell Sci. 2014 Mar 15;127(Pt 6):1169–78.

20.

Aikawa

Whipple

Lopez

Gunn

Young

Lander

Korc

. Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells. J Clin Invest. 2008 Jan;118(1):89–99.

21.

Whipple

Young

Korc

. A KrasG12D-driven genetic mouse model of pancreatic cancer requires glypican-1 for efficient proliferation and angiogenesis. Oncogene. 2012 May 17;31(20):2535–44.

22.

Kleeff

Kayed

Felix

Penzel

Buchler

Korc

Friess

. Glypican-1 antisense transfection modulates TGF-beta-dependent signaling in Colo-357 pancreatic cancer cells. Biochem Biophys Res Commun. 2004 Aug 6;320(4):1148–55.

23.

Kleeff

Ishiwata

Kumbasar

Friess

Buchler

Lander

Korc

. The cell-surface heparan sulfate proteoglycan glypican-1 regulates growth factor action in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer. J Clin Invest. 1998 Nov 1;102(9):1662–73.

24.

Duan

Feng

Lei

. GPC-1 may serve as a predictor of perineural invasion and a prognosticator of survival in pancreatic cancer. Asian J Surg. 2013 Jan;36(1):7–12.

25.

Niu

Liu

Gao

Sun

Zhao

. Elevated glypican-1 expression is associated with an unfavorable prognosis in pancreatic ductal adenocarcinoma. Cancer Med. 2017 Jun;6(6):1181–91.

26.

Ren

Torres

. Chimeric antigen receptor T-cell therapy targeting glypican-1 in pancreatic cancer. Proceedings of the American Association for Cancer Research Annual Meeting; 2019 Mar 29–Apr 3; Atlanta. Philadelphia: American Association for Cancer Research; 2019.

27.

Matsuda

Maruyama

Guo

Kleeff

Itakura

Matsumoto

Lander

Korc

. Glypican-1 is overexpressed in human breast cancer and modulates the mitogenic effects of multiple heparin-binding growth factors in breast cancer cells. Cancer Res. 2001 Jul 15;61(14):5562–9.

28.

Meyer

Nandini

Qiao

Salamat

Friedl

. Glypican-1 is frequently overexpressed in human gliomas and enhances FGF-2 signaling in glioma cells. Am J Pathol. 2006 Jun;168(6):2014–26.

29.

Saito

Sugiyama

Hama

Yamasaki

Takayasu

Nosaka

Onishi

Muragaki

Kawamata

Kurisu

. High expression of glypican-1 predicts dissemination and poor prognosis in glioblastomas. World Neurosurg. 2017 Sep;105:282–88.

30.

Suhovskih

Mostovich

Kunin

Boboev

Nepomnyashchikh

Aidagulova

Grigorieva

. Proteoglycan expression in normal human prostate tissue and prostate cancer. ISRN Oncol. 2013;2013:680136.

31.

Huang

Izzi

Greenspan

. alpha3 chains of type V collagen regulate breast tumour growth via glypican-1. Nat Commun. 2017 Jan 19;8:14351.

32.

Hara

Takahashi

Serada

Fujimoto

Ohkawara

Nakatsuka

Harada

Nishigaki

Takahashi

Nojima

Miyazaki

Makino

Kurokawa

Yamasaki

Miyata

Nakajima

Takiguchi

Morii

Mori

Doki

Naka

. Overexpression of glypican-1 implicates poor prognosis and their chemoresistance in oesophageal squamous cell carcinoma. Br J Cancer. 2016 Jun 28;115(1):66–75.

33.

Matsuzaki

Serada

Hiramatsu

Nojima

Matsuzaki

Ueda

Ohkawara

Mabuchi

Fujimoto

Morii

Yoshino

Kimura

Naka

. Anti-glypican-1 antibody-drug conjugate exhibits potent preclinical antitumor activity against glypican-1 positive uterine cervical cancer. Int J Cancer. 2018 Mar 1;142(5):1056–66.

34.

Tanaka

Ishikawa

Ushiku

Morikawa

Isagawa

Yamagishi

Yamamoto

Katoh

Takeshita

Arita

Sakamoto

Hasegawa

Kokudo

Fukayama

. EVI1 modulates oncogenic role of GPC1 in pancreatic carcinogenesis. Oncotarget. 2017 Nov 21;8(59):99552–66.

35.

Melo

Luecke

Kahlert

Fernandez

Gammon

Kaye

LeBleu

Mittendorf

Weitz

Rahbari

Reissfelder

Pilarsky

Fraga

Piwnica-Worms

Kalluri

. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature. 2015 Jul 9;523(7559):177–82.

36.

Lai

Wang

McElyea

Sherman

House

Korc

. A microRNA signature in circulating exosomes is superior to exosomal glypican-1 levels for diagnosing pancreatic cancer. Cancer Lett. 2017 May 1;393:86–93.

37.

Frampton

Prado

Lopez-Jimenez

Fajardo-Puerta

Jawad

ZAR

Lawton

Giovannetti

Habib

Castellano

Stebbing

Krell

Jiao

. Glypican-1 is enriched in circulating-exosomes in pancreatic cancer and correlates with tumor burden. Oncotarget. 2018 Apr 10;9(27):19006–13.

38.

Babina

Turner

. Advances and challenges in targeting FGFR signalling in cancer. Nat Rev Cancer. 2017 May;17(5):318–32.

39.

Olsson

Dimberg

Kreuger

Claesson—Welsh

. VEGF receptor signalling—in control of vascular function. Nat Rev Mol Cell Biol. 2006 May;7(5):359–71.

40.

Kayed

Kleeff

Keleg

Jiang

Penzel

Giese

Zentgraf

Buchler

Korc

Friess

. Correlation of glypican-1 expression with TGF-beta, BMP, and activin receptors in pancreatic ductal adenocarcinoma. Int J Oncol. 2006 Nov;29(5):1139–48.

41.

Harada

Serada

Fujimoto

Takahashi

Hara

Nakatsuka

Sugase

Nishigaki

Saito

Hiramatsu

Nojima

Mitsuo

Ohkawara

Morii

Mori

Doki

Kaneda

Naka

. Glypican-1 targeted antibody-based therapy induces preclinical antitumor activity against esophageal squamous cell carcinoma. Oncotarget. 2017 Apr 11;8(15):24741–52.

42.

Chen

Zhan

Zhu

Weng

Dong

Liu

Shen

. Glypican-1 promotes tumorigenesis by regulating the PTEN/Akt/beta-catenin signaling pathway in esophageal squamous cell carcinoma. Dig Dis Sci. 2019 Jun;64(6):1493–502.

43.

Nishigaki

Takahashi

Serada

Fujimoto

Ohkawara

Hara

Sugase

Otsuru

Saito

Tsujii

Nomura

Tanaka

Miyazaki

Makino

Kurokawa

Nakajima

Eguchi

Yamasaki

Mori

Doki

Naka

. Anti-glypican-1 antibody-drug conjugate is a potential therapy against pancreatic cancer. Br J Cancer. 2020 Apr;122(9):1333–41.

44.

Porter

Levine

Kalos

Bagg

June

. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011 Aug 25;365(8):725–33.

45.

Kochenderfer

Dudley

Feldman

Wilson

Spaner

Maric

Stetler-Stevenson

Phan

Hughes

Sherry

Yang

Kammula

Devillier

Carpenter

Nathan

Morgan

Laurencot

Rosenberg

. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012 Mar 22;119(12):2709–20.

46.

Kato

Yaguchi

Iwata

Katoh

Morii

Tsubota

Takise

Tamiya

Kamada

Akiba

Tsumoto

Serada

Naka

Nishimura

Nakagawa

Kawakami

. GPC1 specific CAR-T cells eradicate established solid tumor without adverse effects and synergize with anti-PD-1 Ab. Elife. 2020 Mar 31;9:e49392.

47.

Stipp

Litwack

Lander

. Cerebroglycan: an integral membrane heparan sulfate proteoglycan that is unique to the developing nervous system and expressed specifically during neuronal differentiation. J Cell Biol. 1994 Jan;124(1–2):149–60.

48.

Ivins

Litwack

Kumbasar

Stipp

Lander

. Cerebroglycan, a developmentally regulated cell-surface heparan sulfate proteoglycan, is expressed on developing axons and growth cones. Dev Biol. 1997;184:320–32.

49.

Stipp

Litwack

Lander

50.

Orentas

Yang

Wen

Wei

Mackall

Khan

. Identification of cell surface proteins as potential immunotherapy targets in 12 pediatric cancers. Front Oncol. 2012;2:194.

51.

Fleming

. High affinity monoclonal antibodies targeting glypican-2 and uses thereof. 2019. Available from: https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020033430

52.

Maris

Hogarty

Bagatell

Cohn

. Neuro-blastoma. Lancet. 2007;369(9579):2106–20.

53.

Kool

Korshunov

Remke

Jones

Schlanstein

Northcott

Cho

Koster

Schouten-van Meeteren

van Vuurden

Clifford

Pietsch

von Bueren

Rutkowski

McCabe

Collins

Backlund

Haberler

Bourdeaut

Delattre

Doz

Ellison

Gilbertson

Pomeroy

Taylor

Lichter

Pfister

. Molecular subgroups of medulloblastoma: an international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3, and Group 4 medulloblastomas. Acta Neuropathol. 2012 Apr;123(4):473–84.

54.

Jia

Bates

Basom

Eberhart

MacPherson

. A mouse model of MYCN-driven retinoblastoma reveals MYCN-independent tumor reemergence. J Clin Invest. 2017 Mar 1;127(3):888–98.

55.

Shu

Guttentag

Wang

Andl

Ballard

Piccolo

Birchmeier

Whitsett

Millar

Morrisey

. Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung. Dev Biol. 2005 Jul 1;283(1):226–39.

56.

Majzner

Mackall

. Tumor antigen escape from CAR T-cell therapy. Cancer Discov. 2018 Oct;8(10):1219–26.

57.

. Advances in liver cancer antibody therapies: a focus on glypican-3 and mesothelin. BioDrugs. 2011 Oct 1;25(5):275–84.

58.

Iglesias

Centeno

Pascuccelli

Ward

Peters

Filmus

Puricelli

de Kier Joffe

. Expression pattern of glypican-3 (GPC3) during human embryonic and fetal development. Histol Histopathol. 2008 Nov;23(11):1333–40.

59.

Capurro

Xiang

Lobe

Filmus

. Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling. Cancer Res. 2005 Jul 15;65(14):6245–54.

60.

Gao

Tang

Zhang

Feng

Qian

Dimitrov

. Immunotoxin targeting glypican-3 regresses liver cancer via dual inhibition of Wnt signalling and protein synthesis. Nat Commun. 2015 Mar 11;6:6536.

61.

Capurro

Shi

Izumikawa

Kitagawa

Filmus

. Processing by convertases is required for glypican-3-induced inhibition of Hedgehog signaling. J Biol Chem. 2015 Mar 20;290(12):7576–85.

62.

Capurro

Shi

Jia

Filmus

. Glypican-3 inhibits Hedgehog signaling during development by competing with patched for Hedgehog binding. Dev Cell. 2008 May;14(5):700–11.

63.

Filmus

Capurro

. The role of glypicans in Hedgehog signaling. Matrix Biol. 2014 Apr;35:248–52.

64.

Gao

Kim

. Human monoclonal antibody targeting the heparan sulfate chains of glypican-3 inhibits HGF-mediated migration and motility of hepatocellular carcinoma cells. PLoS ONE. 2015;10(9):e0137664.

65.

Hsu

Cheng

Lai

. Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellular carcinoma: biological significance and temporospatial distribution. Cancer Res. 1997 Nov 15;57(22):5179–84.

66.

Capurro

Wanless

Sherman

Deboer

Shi

Miyoshi

Filmus

. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology. 2003 Jul;125(1):89–97.

67.

Baumhoer

Tornillo

Stadlmann

Roncalli

Diamantis

Terracciano

. Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples. Am J Clin Pathol. 2008 Jun;129(6):899–906.

68.

Zhu

Friess

Wang

Abou-Shady

Zimmermann

Lander

Korc

Kleeff

Buchler

. Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders. Gut. 2001 Apr;48(4):558–64.

69.

Libbrecht

Severi

Cassiman

Vander Borght

Pirenne

Nevens

Verslype

van Pelt

Roskams

. Glypican-3 expression distinguishes small hepatocellular carcinomas from cirrhosis, dysplastic nodules, and focal nodular hyperplasia-like nodules. Am J Surg Pathol. 2006 Nov;30(11):1405–11.

70.

Llovet

Chen

Wurmbach

Roayaie

Fiel

Schwartz

Thung

Khitrov

Zhang

Villanueva

Battiston

Mazzaferro

Bruix

Waxman

Friedman

. A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis. Gastroenterology. 2006 Dec;131(6):1758–67.

71.

Kaseb

Hassan

Lacin

Abdel-Wahab

Amin

Shalaby

Wolff

Yao

Rashid

Vennapusa

Feng

Ohtomo

. Evaluating clinical and prognostic implications of Glypican-3 in hepatocellular carcinoma. Oncotarget. 2016 Oct 25;7(43):69916–26.

72.

Liu

Yang

Zeng

. Prognostic significance of glypican-3 expression in hepatocellular carcinoma: a meta-analysis. Medicine (Baltimore). 2018 Jan;97(4):e9702.

73.

Shirakawa

Suzuki

Shimomura

Kojima

Gotohda

Takahashi

Nakagohri

Konishi

Kobayashi

Kinoshita

Nakatsura

. Glypican-3 expression is correlated with poor prognosis in hepatocellular carcinoma. Cancer Sci. 2009 Aug;100(8):1403–7.

74.

Zhang

Song

. Overexpression of glypican-3 is a predictor of poor prognosis in hepatocellular carcinoma: an updated meta-analysis. Medicine (Baltimore). 2018 Jun;97(24):e11130.

75.

Takai

Kato

Watanabe

Matsubara

Suzuki

Kataoka

. The expression profile of glypican-3 and its relation to macrophage population in human hepatocellular carcinoma. Liver Int. 2009 Aug;29(7):1056–64.

76.

Phung

Gao

Man

Nagata

. High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening. MABs. 2012 Sep–Oct;4(5):592–9.

77.

Aviel-Ronen

Lau

Pintilie

Lau

Liu

Tsao

Jothy

. Glypican-3 is overexpressed in lung squamous cell carcinoma, but not in adenocarcinoma. Mod Pathol. 2008 Jul;21(7):817–25.

78.

Nakatsura

Kageshita

Ito

Wakamatsu

Monji

Ikuta

Senju

Ono

Nishimura

. Identification of glypican-3 as a novel tumor marker for melanoma. Clin Cancer Res. 2004 Oct 1;10(19):6612–21.

79.

Fang

Liu

Weiss

Chu

. Frequent expression of glypican-3 in Merkel cell carcinoma: an immunohistochemical study of 55 cases. Appl Immuno-histochem Mol Morphol. 2009 Jan;17(1):40–6.

80.

Maeda

Ota

Takazawa

Aburatani

Nakagawa

Yano

Taketani

Kodama

Fukayama

. Glypican-3 expression in clear cell adenocarcinoma of the ovary. Mod Pathol. 2009 Jun;22(6):824–32.

81.

Yamanaka

Ito

Okuyama

Noda

Matsumoto

Yoshida

Miyauchi

Capurro

Filmus

Miyoshi

. Immunohistochemical study of glypican 3 in thyroid cancer. Oncology. 2007;73(5–6):389–94.

82.

Aydin

Yildiz

Baris

Dundar

Karagoz

. Expression of glypican 3 in low and high grade urothelial carcinomas. Diagn Pathol. 2015 Apr 21;10:34.

83.

Andisheh-Tadbir

Ashraf

Gudarzi

Zare

. Evaluation of glypican-3 expression in benign and malignant salivary gland tumors. J Oral Biol Craniofac Res. 2019 Jan–Mar;9(1):63–6.

84.

Moek

Fehrmann

RSN

van der Vegt

de Vries

EGE

de Groot

DJA

. Glypican 3 overexpression across a broad spectrum of tumor types discovered with functional genomic mRNA profiling of a large cancer database. Am J Pathol. 2018 Sep;188(9):1973–81.

85.

Lin

Hatcher

Lozzi

Kong

Huang-Hobbs

Cheng

Beechar

Zhu

Zhang

Chen

Mills

Mohila

Creighton

Noebels

Scott

Deneen

. PIK3CA variants selectively initiate brain hyperactivity during gliomagenesis. Nature. 2020 Feb;578(7793):166–71.

86.

Toretsky

Zitomersky

Eskenazi

Voigt

Strauch

Sun

Huber

Meltzer

Schlessinger

. Glypican-3 expression in Wilms tumor and hepatoblastoma. J Pediatr Hematol Oncol. 2001 Nov;23(8):496–9.

87.

Yamauchi

Watanabe

Hishinuma

Ohashi

Midorikawa

Morishita

Niki

Shibahara

Mori

Makuuchi

Hippo

Kodama

Iwanari

Aburatani

Fukayama

. The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod Pathol. 2005 Dec;18(12):1591–8.

88.

Zynger

Gupta

Luan

Chou

Yang

. Expression of glypican 3 in hepatoblastoma: an immunohistochemical study of 65 cases. Hum Pathol. 2008 Feb;39(2):224–30.

89.

Chan

Pawel

Corao

Venneti

Russo

Santi

Sullivan

. Immunohistochemical expression of glypican-3 in pediatric tumors: an analysis of 414 cases. Pediatr Dev Pathol. 2013 Jul–Aug;16(4):272–7.

90.

Kinoshita

Tanaka

Souzaki

Miyoshi

Kohashi

Oda

Nakatsura

Taguchi

. Glypican 3 expression in pediatric malignant solid tumors. Eur J Pediatr Surg. 2015 Feb;25(1):138–44.

91.

Esheba

Pate

Longacre

. Oncofetal protein glypican-3 distinguishes yolk sac tumor from clear cell carcinoma of the ovary. Am J Surg Pathol. 2008 Apr;32(4):600–7.

92.

Zynger

Dimov

Luan

Teh

Yang

. Glypican 3: a novel marker in testicular germ cell tumors. Am J Surg Pathol. 2006 Dec;30(12):1570–5.

93.

Zynger

Everton

Dimov

Chou

Yang

. Expression of glypican 3 in ovarian and extragonadal germ cell tumors. Am J Clin Pathol. 2008 Aug;130(2):224–30.

94.

Saikali

Sinnett

. Expression of glypican 3 (GPC3) in embryonal tumors. Int J Cancer. 2000 Sep 20;89(5):418–22.

95.

Tretiakova

Zynger

Luan

Andeen

Finn

Kocherginsky

Teh

Yang

. Glypican 3 overexpression in primary and metastatic Wilms tumors. Virchows Arch. 2015 Jan;466(1):67–76.

96.

White

Kelsey

Varley

Birch

. Somatic glypican 3 (GPC3) mutations in Wilms’ tumour. Br J Cancer. 2002 Jun 17;86(12):1920–2.

97.

Thway

Selfe

Missiaglia

Fisher

Shipley

. Glypican-3 is expressed in rhabdomyosarcomas but not adult spindle cell and pleomorphic sarcomas. J Clin Pathol. 2011 Jul;64(7):587–91.

98.

Pilia

Hughes-Benzie

MacKenzie

Baybayan

Chen

Huber

Neri

Cao

Forabosco

Schlessinger

. Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-Behmel overgrowth syndrome. Nature Genetics. 1996;12(3):241–47.

99.

Cano-Gauci

Song

Yang

McKerlie

Choo

Shi

Pullano

Piscione

Grisaru

Soon

Sedlackova

Tanswell

Mak

Yeger

Lockwood

Rosenblum

Filmus

. Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome. J Cell Biol. 1999 Jul 12;146(1):255–64.

100.

Murthy

Shen

De Rienzo

Lee

Ferriola

Jhanwar

Mossman

Filmus

Testa

. Expression of GPC3, an X-linked recessive overgrowth gene, is silenced in malignant mesothelioma. Oncogene. 2000 Jan 20;19(3):410–6.

101.

Valsechi

Oliveira

Conceicao

Stuqui

Candido

Provazzi

de Araujo

Silva

Jr de Freitas Calmon

Rahal

. GPC3 reduces cell proliferation in renal carcinoma cell lines. BMC Cancer. 2014 Aug 29;14:631.

102.

Xiang

Ladeda

Filmus

. Glypican-3 expression is silenced in human breast cancer. Oncogene. 2001 Nov 1;20(50):7408–12.

103.

Lin

Huber

Schlessinger

Morin

. Frequent silencing of the GPC3 gene in ovarian cancer cell lines. Cancer Res. 1999 Feb 15;59(4):807–10.

104.

Kim

Borczuk

Busch

Filmus

Capurro

Brody

Lange

D’Armiento

Rothman

Powell

. The heparan sulfate proteoglycan GPC3 is a potential lung tumor suppressor. Am J Respir Cell Mol Biol. 2003 Dec;29(6):694–701.

105.

Trinh

Puszyk

Liu

. Epigenetic regulation of glypican-3 in hepatocellular carcinoma. Cancer Res. 2014;74(Suppl 19):418.

106.

Jin

Zhang

Liu

Chen

. Oncogenic activation of glypican-3 by c-Myc in human hepatocellular carcinoma. Hepatology. 2012 Oct;56(4):1380–90.

107.

Jalvy-Delvaille

Maurel

Majo

Pierre

Chabas

Combe

Rosenbaum

Sagliocco

Grosset

. Molecular basis of differential target regulation by miR-96 and miR-182: the Glypican-3 as a model. Nucleic Acids Res. 2012 Feb;40(3):1356–65.

108.

Maurel

Jalvy

Ladeiro

Combe

Vachet

Sagliocco

Bioulac-Sage

Pitard

Jacquemin-Sablon

Zucman-Rossi

Laloo

Grosset

. A functional screening identifies five microRNAs controlling glypican-3: role of miR-1271 down-regulation in hepatocellular carcinoma. Hepatology. 2013 Jan;57(1):195–204.

109.

Bengochea

de Souza

Lefrancois

Le Roux

Galy

Chemin

Kim

Wands

Trepo

Hainaut

Scoazec

Vitvitski

Merle

. Common dysregulation of Wnt/Frizzled receptor elements in human hepatocellular carcinoma. Br J Cancer. 2008 Jul 8;99(1):143–50.

110.

Gao

Liu

. Epitope mapping by a Wnt-blocking antibody: evidence of the Wnt binding domain in heparan sulfate. Sci Rep. 2016 May 17;6:26245.

111.

Midorikawa

Ishikawa

Iwanari

Imamura

Sakamoto

Miyazono

Kodama

Makuuchi

Aburatani

. Glypican-3, overexpressed in hepatocellular carcinoma, modulates FGF2 and BMP-7 signaling. Int J Cancer. 2003 Feb 10;103(4):455–65.

112.

Song

Shi

Filmus

. OCI-5/rat glypican-3 binds to fibroblast growth factor-2 but not to insulin-like growth factor-2. J Biol Chem. 1997 Mar 21;272(12):7574–7.

113.

Sun

Chua

. Suppression of glypican 3 inhibits growth of hepatocellular carcinoma cells through up-regulation of TGF-beta2. Neoplasia. 2011 Aug;13(8):735–47.

114.

Capurro

Filmus

. Overgrowth of a mouse model of Simpson-Golabi-Behmel syndrome is partly mediated by Indian hedgehog. EMBO Rep. 2009 Aug;10(8):901–7.

115.

Filmus

Capurro

. The role of glypican-3 in the regulation of body size and cancer. Cell Cycle. 2008 Sep 15;7(18):2787–90.

116.

Capurro

Shi

Filmus

. LRP1 mediates Hedgehog-induced endocytosis of the GPC3-Hedgehog complex. J Cell Sci. 2012 Jul 15;125(Pt 14):3380–9.

117.

Wolfe

Septer

Edwards

Zhong

Abdulkarim

Ranganathan

Apte

. Deregulation of Hippo kinase signalling in human hepatic malignancies. Liver Int. 2012 Jan;32(1):38–47.

118.

Filmus

. Glypicans in growth control and cancer. Glycobiology. 2001 Mar;11(3):19R–23R.

119.

Chen

Horiuchi

Kikuchi

Osada

Yoshida

Shiozawa

Konishi

. Hedgehog signal pathway is activated in ovarian carcinomas, correlating with cell proliferation: it’s inhibition leads to growth suppression and apoptosis. Cancer Sci. 2007 Jan;98(1):68–76.

120.

Kubo

Nakamura

Tasaki

Yamanaka

Nakashima

Nomura

Kuroki

Katano

. Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer. Cancer Res. 2004 Sep 1;64(17):6071–4.

121.

Komori

Nakatsura

Senju

Yoshitake

Motomura

Ikuta

Fukuma

Yokomine

Harao

Beppu

Matsui

Torigoe

Sato

Baba

Nishimura

. Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma. Clin Cancer Res. 2006 May 1;12(9):2689–97.

122.

Nakatsura

Komori

Kubo

Yoshitake

Senju

Katagiri

Furukawa

Ogawa

Nakamura

Nishimura

. Mouse homologue of a novel human oncofetal antigen, glypican-3, evokes T-cell-mediated tumor rejection without autoimmune reactions in mice. Clin Cancer Res. 2004 Dec 15;10(24):8630–40.

123.

Sawada

Yoshikawa

Nobuoka

Shirakawa

Kuronuma

Motomura

Mizuno

Ishii

Nakachi

Konishi

Nakagohri

Takahashi

Gotohda

Takayama

Yamao

Uesaka

Furuse

Kinoshita

Nakatsura

. Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival. Clin Cancer Res. 2012 Jul 1;18(13):3686–96.

124.

Yoshikawa

Nakatsugawa

Suzuki

Shirakawa

Nobuoka

Sakemura

Motomura

Tanaka

Hayashi

Nakatsura

. HLA-A2-restricted glypican-3 peptide-specific CTL clones induced by peptide vaccine show high avidity and antigen-specific killing activity against tumor cells. Cancer Sci. 2011 May;10 2(5):918–25.

125.

Sawada

Yoshikawa

Ofuji

Yoshimura

Tsuchiya

Takahashi

Nobuoka

Gotohda

Takahashi

Kato

Konishi

Kinoshita

Ikeda

Nakachi

Yamazaki

Mizuno

Takayama

Yamao

Uesaka

Furuse

Endo

Nakatsura

. Phase II study of the GPC3-derived peptide vaccine as an adjuvant therapy for hepatocellular carcinoma patients. Oncoimmunology. 2016 May;5(5):e1129483.

126.

Sayem

Tomita

Yuno

Hirayama

Irie

Tsukamoto

Senju

Yuba

Yoshikawa

Kono

Nakatsura

Nishimura

. Identification of glypican-3-derived long peptides activating both CD8(+) and CD4(+) T cells; prolonged overall survival in cancer patients with Th cell response. Oncoimmunology. 2016;5(1):e1062209.

127.

Suzuki

Sakata

Utsumi

Sekiya

Kajiyama

Shibata

Kikkawa

Nakatsura

. Efficacy of glypican-3-derived peptide vaccine therapy on the survival of patients with refractory ovarian clear cell carcinoma. Oncoimmunology. 2016;5(11):e1238542.

128.

Tsuchiya

Hosono

Yoshikawa

Shoda

Nosaka

Shimomura

Hara

Nitani

Manabe

Yoshihara

Hosoya

Kaneda

Kinoshita

Kohashi

Yoshimura

Fujinami

Saito

Mizuno

Nakatsura

. Phase I study of glypican-3-derived peptide vaccine therapy for patients with refractory pediatric solid tumors. Oncoimmunology. 2017;7(1):e1377872.

129.

Ishiguro

Sugimoto

Kinoshita

Miyazaki

Nakano

Tsunoda

Sugo

Ohizumi

Aburatani

Hamakubo

Kodama

Tsuchiya

Yamada-Okabe

. Anti-glypican 3 antibody as a potential antitumor agent for human liver cancer. Cancer Res. 2008 Dec 1;68(23):9832–8.

130.

Nakano

Orita

Nezu

Yoshino

Ohizumi

Sugimoto

Furugaki

Kinoshita

Ishiguro

Hamakubo

Kodama

Aburatani

Yamada-Okabe

Tsuchiya

. Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells. Biochem Biophys Res Commun. 2009 Jan 9;378(2):279–84.

131.

Zhu

Gold

El-Khoueiry

Abrams

Morikawa

Ohishi

Ohtomo

Philip

. First-in-man phase I study of GC33, a novel recombinant humanized antibody against glypican-3, in patients with advanced hepatocellular carcinoma. Clin Cancer Res. 2013 Feb 15;19(4):920–8.

132.

Yen

C-J

Daniele

Kudo

Merle

Park

J-W

Ross

Peron

J-M

Ebert

Chan

Poon

RTP

Colombo

Okusaka

Ryoo

B-Y

Minguez

Tanaka

Ohtomo

Rutman

Chen

Y-C

Lee

R-M

Abou-Alfa

. Randomized phase II trial of intravenous RO5137382/GC33 at 1600 mg every other week and placebo in previously treated patients with unresectable advanced hepatocellular carcinoma (HCC; NCT01507168). J Clin Oncol. 2014;32(Suppl 15):4102.

133.

Zhang

Feng

Schneider

Zhou

Mackay

Kramer

Duan

Yang

Kolluri

Hummer

Torres

Zhu

Hall

Luo

Chen

Wang

Abate-Daga

Dropublic

Hewitt

Orentas

Greten

Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology. 2020 Jun;158(8):2250–65.

134.

Zhang

. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma. Sci Rep. 2016 Sep 26;6:33878.

135.

Kim

. Isolation of antibodies to heparan sulfate on glypicans by phage display. Curr Protoc Protein Sci. 2018 Nov;94(1):e66.

136.

Wang

Gao

Feng

Pastan

. Construction of an immunotoxin, HN3-mPE24, targeting glypican-3 for liver cancer therapy. Oncotarget. 2017 May 16;8(20):32450–60.

137.

Fleming

Urban

Hall

Longerich

Greten

Pastan

. Engineered anti-GPC3 immunotoxin, HN3-ABD-T20, produces regression in mouse liver cancer xenografts through prolonged serum retention. Hepatology. 2020 May;71(5):1696–711.

138.

Gao

Pan

Jiang

Shi

Kong

Wang

Yang

. Development of T cells redirected to glypican-3 for the treatment of hepatocellular carcinoma. Clin Cancer Res. 2014 Dec 15;20(24):6418–28.

139.

Guo

Rathi

Marinova

Gao

Liu

Dotti

Gottschalk

Metelitsa

Heczey

. Redirecting T cells to glypican-3 with 4-1BB zeta chimeric antigen receptors results in Th1 polarization and potent antitumor activity. Hum Gene Ther. 2017 May;28(5):437–48.

140.

Jiang

Chen

Lai

Wei

Lin

Wang

Liang

Liu

Peng

Weng

Pei

Liu

Yao

Xue

. Anti-GPC3-CAR T cells suppress the growth of tumor cells in patient-derived xenografts of hepatocellular carcinoma. Front Immunol. 2016;7:690.

141.

Wang

Qin

Liu

Jiang

Cui

Liu

Fang

Tang

Jin

Qian

. PiggyBac-engineered T cells expressing a glypican-3-specific chimeric antigen receptor show potent activities against hepatocellular carcinoma. Immunobiology. 2020 Jan;225(1):151850.

142.

Guo

Jiang

Shi

Zhou

Zhang

Shi

Luo

Wang

Sun

. Disruption of PD-1 enhanced the anti-tumor activity of chimeric antigen receptor T cells against hepatocellular carcinoma. Front Pharmacol. 2018;9:1118.

143.

Pan

Shi

Jiang

Shi

Liu

Wang

Luo

. Increased antitumor activities of glypican-3-specific chimeric antigen receptor-modified T cells by coexpression of a soluble PD1-CH3 fusion protein. Cancer Immunol Immunother. 2018 Oct;67(10):1621–34.

144.

Shi

Kaseb

Zhang

Chi

Gao

Jiang

Wang

Yuan

Wang

Zhai

Chimeric antigen receptor-glypican-3 T-cell therapy for advanced hepatocellular carcinoma: Results of phase 1 trials. Clin Cancer Res. 2020 May 5.

145.

Urban

Nani

Zhang

Shah

Gorka

Guha

Chen

Hall

Schnermann

. Glypican-3-specific antibody drug conjugates targeting hepatocellular carcinoma. Hepatology. 2019 Aug;70(2):563–76.

146.

Singh

Settleman

. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene. 2010 Aug 26;29(34):4741–51.

147.

Xiong

Sun

Zhang

Zhu

Zhuang

Zhang

Wang

Qin

Tang

. Human hepatocellular carcinoma tumor-derived endothelial cells manifest increased angiogenesis capability and drug resistance compared with normal endothelial cells. Clin Cancer Res. 2009 Aug 1;15(15):4838–46.

148.

Lipovsek

Carvajal

Allentoff

Barros

Jr. Brailsford

Cong

Cotter

Gangwar

Hollander

Lafont

Lau

Moreta

O’Neil

Pinckney

Smith

Terragni

Wallace

Wang

Wright

Marsh

Bryson

. Adnectin-drug conjugates for Glypican-3-specific delivery of a cytotoxic payload to tumors. Protein Eng Des Sel. 2018 May 1;31(5):159–71.

149.

Hanaoka

Nagaya

Sato

Nakamura

Watanabe

Harada

Gao

Feng

Phung

Kim

Paik

Choyke

Kobayashi

. Glypican-3 targeted human heavy chain antibody as a drug carrier for hepatocellular carcinoma therapy. Mol Pharm. 2015 Jun 1;12(6):2151–7.

150.

Hanaoka

Nakajima

Sato

Watanabe

Phung

Gao

Harada

Kim

Paik

Choyke

Kobayashi

. Photoimmunotherapy of hepatocellular carcinoma-targeting glypican-3 combined with nanosized albumin-bound paclitaxel. Nanomedicine (Lond). 2015;10(7):1139–47.

151.

Ishiguro

Sano

Komatsu

Kamata-Sakurai

Kaneko

Kinoshita

Shiraiwa

Azuma

Tsunenari

Kayukawa

Sonobe

Ono

Sakata

Fujii

Miyazaki

Noguchi

Endo

Harada

Frings

Fujii

Nanba

Narita

Sakamoto

Wakabayashi

Konishi

Segawa

Igawa

Tsushima

Mutoh

Nishito

Takahashi

Stewart

ElGabry

Kawabe

Ishigai

Chiba

Aoki

Hattori

Nezu

. An anti-glypican 3/CD3 bispecific T cell-redirecting antibody for treatment of solid tumors. Sci Transl Med. 2017 Oct 4;9(410):eaal4291.

152.

Jiang

Wang

Song

Wang

Kong

. Treatment of hepatocellular carcinoma with a GPC3-targeted bispecific T cell engager. Oncotarget. 2017 Aug 8;8(32):52866–76.

153.

Dargel

Bassani-Sternberg

Hasreiter

Zani

Bockmann

Thiele

Bohne

Wisskirchen

Wilde

Sprinzl

Schendel

Krackhardt

Uckert

Wohlleber

Schiemann

Stemmer

Heikenwalder

Busch

Richter

Mann

Protzer

. T cells engineered to express a T-cell receptor specific for glypican-3 to recognize and kill hepatoma cells in vitro and in mice. Gastroenterology. 2015 Oct;149(4):1042–52.

154.

Luo

Fan

Shi

Liu

Pan

Jiang

. Development of GPC3-specific chimeric antigen receptor-engineered natural killer cells for the treatment of hepatocellular carcinoma. Mol Ther. 2018 Feb 7;26(2):366–78.

155.

Veugelers

Vermeesch

Watanabe

Yamaguchi

Marynen

David

. GPC4, the gene for human K-glypican, flanks GPC3 on xq26: deletion of the GPC3-GPC4 gene cluster in one family with Simpson-Golabi-Behmel syndrome. Genomics. 1998 Oct 1;53(1):1–11.

156.

Watanabe

Yamada

Yamaguchi

. K-glypican: a novel GPI-anchored heparan sulfate proteoglycan that is highly expressed in developing brain and kidney. J Cell Biol. 1995 Sep;130(5):1207–18.

157.

Cao

Sun

Qin

Zhou

Cheng

Chen

Qian

Duan

Wang

Han

. Targeting glypican-4 overcomes 5-FU resistance and attenuates stem cell-like properties via suppression of Wnt/beta-catenin pathway in pancreatic cancer cells. J Cell Biochem. 2018 Nov;119(11):9498–512.

158.

Fang

Shen

Zhan

Feng

Chen

Deng

Pan

Ding

. CD36 inhibits beta-catenin/c-myc-mediated glycolysis through ubiquitination of GPC4 to repress colorectal tumorigenesis. Nat Commun. 2019 Sep 4;10(1):3981.

159.

Zhao

Liu

Sun

Zhao

Liu

Kuang

Shu

Luo

. Glypican-4 gene polymorphism (rs1048369) and susceptibility to Epstein-Barr virus-associated and -negative gastric carcinoma. Virus Res. 2016 Jul 15;220:52–6.

160.

Liu

Zhao

Zhang

Zhao

Luo

. The glypican-4 gene polymorphism rs1048369 and susceptibility to Epstein-Barr virus-positive and -negative nasopharyngeal carcinoma in Northern China. Oncol Res Treat. 2019;42(11):572–9.

161.

Varma

Hector

Clark

Greco

Hawthorn

Pendyala

. Gene expression profiling of a clonal isolate of oxaliplatin-resistant ovarian carcinoma cell line A2780/C10. Oncol Rep. 2005 Oct;14(4):925–32.

162.

Munir

Van Ngu

Na Ayudthaya

Ryu

. Downregulation of glypican-4 facilitates breast cancer progression by inducing cell migration and proliferation. Biochem Biophys Res Commun. 2020 May 21;526(1):91–7.

163.

Waterson

Stockley

Segal

Golabi

. Novel duplication in glypican-4 as an apparent cause of Simpson-Golabi-Behmel syndrome. Am J Med Genet A. 2010 Dec;152A(12):3179–81.

164.

Sakane

Yamamoto

Matsumoto

Sato

Kikuchi

. Localization of glypican-4 in different membrane microdomains is involved in the regulation of Wnt signaling. J Cell Sci. 2012 Jan 15;125(Pt 2):449–60.

165.

Silverstein

Febbraio

. CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior. Sci Signal. 2009 May 26;2(72):re3.

166.

Lebensohn

Rohatgi

. R-spondins can potentiate WNT signaling without LGRs. Elife. 2018 Feb 6;7:e33126.

167.

Dubey

van Kerkhof

Jordens

Malinauskas

Pusapati

McKenna

Carette

Siebold

Maurice

Lebensohn

Rohatgi

R-spondins engage heparan sulfate proteoglycans to potentiate WNT signaling. Elife. 2020 May 20;9.

168.

Saunders

Paine-Saunders

Lander

. Expression of the cell surface proteoglycan glypican-5 is developmentally regulated in kidney, limb, and brain. Dev Biol. 1997 Oct 1;190(1):78–93.

169.

Veugelers

Vermeesch

Reekmans

Steinfeld

Marynen

David

. Characterization of glypican-5 and chromosomal localization of human GPC5, a new member of the glypican gene family. Genomics. 1997 Feb 15;40(1):24–30.

170.

Williamson

Selfe

Gordon

Pritchard-Jones

Murai

Jones

Workman

Shipley

. Role for amplification and expression of glypican-5 in rhabdomyosarcoma. Cancer Res. 2007 Jan 1;67(1):57–65.

171.

Nishimura

Takita

Sato-Otsubo

Kato

Koh

Hanada

Tanaka

Kato

Maeda

Fukayama

Sanada

Hayashi

Ogawa

. Characterization of genetic lesions in rhabdomyosarcoma using a high-density single nucleotide polymorphism array. Cancer Sci. 2013 Jul;104(7):856–64.

172.

Sheu

de Andrade

Wang

Chang

Aubry

Aakre

Allen

Chen

Cunningham

Deschamps

Jiang

Lin

Marks

Pankratz

Sun

Tang

Vasmatzis

Harris

Spitz

Jen

Wang

Zhang

Christiani

Yang

. Genetic variants and risk of lung cancer in never smokers: a genome-wide association study. Lancet Oncol. 2010 Apr;11(4):321–30.

173.

Yang

Zhang

Qiu

Fan

Wang

Yin

. Glypican-5 is a novel metastasis suppressor gene in non-small cell lung cancer. Cancer Lett. 2013 Dec 1;341(2):265–73.

174.

Yuan

Liu

Zhang

Xiang

Cai

Zhang

Liao

Wang

Yang

Bai

. GPC5, a novel epigenetically silenced tumor suppressor, inhibits tumor growth by suppressing Wnt/beta-catenin signaling in lung adenocarcinoma. Oncogene. 2016 Nov 24;35(47):6120–31.

175.

Miao

Cai

Ding

Xiao

Yang

Zhang

. The overexpression of glypican-5 promotes cancer cell migration and is associated with shorter overall survival in non-small cell lung cancer. Oncol Lett. 2013 Dec;6(6):1565–72.

176.

Zhang

Liu

. A lung cancer gene GPC5 could also be crucial in breast cancer. Mol Genet Metab. 2011 May;10 3(1):104–5.

177.

Zhang

Liu

Wang

Qiao

Zhang

. Prognostic significance of GPC5 expression in patients with prostate cancer. Tumour Biol. 2016 May;37(5):6413–8.

178.

Yuan

Zhang

Cao

Yang

. High expression of microRNA-4295 contributes to cell proliferation and invasion of pancreatic ductal adenocarcinoma by the down-regulation of Glypican-5. Biochem Biophys Res Commun. 2018 Feb 26;497(1):73–9.

179.

Liu

Zhang

Sha

Liu

Zhang

Wang

. miR-709 up-regulated in hepatocellular carcinoma, promotes proliferation and invasion by targeting GPC5. Cell Prolif. 2015 Jun;48(3):330–7.

180.

Hong

Zhang

Pan

Zhai

Zhang

Huang

Pei

Liu

. MicroRNA-301b promotes the proliferation and invasion of glioma cells through enhancing activation of Wnt/beta-catenin signaling via targeting Glypican-5. Eur J Pharmacol. 2019 Jul 5;854:39–47.

181.

Shi

Capurro

Filmus

. Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling. J Cell Biol. 2011 Feb 21;192(4):691–704.

182.

Wang

Qiu

Xia

Mao

Wang

Dong

Yang

Yin

. Glypican-5 suppresses Epithelial-Mesenchymal Transition of the lung adenocarcinoma by competitively binding to Wnt3a. Oncotarget. 2016 Nov 29;7(48):79736–46.

183.

Wang

Liu

Guan

. Hypoxia-responsive mir-301a and mir-301b promote radioresistance of prostate cancer cells via downregulating NDRG2. Med Sci Monit. 2016 Jun 21;22:2126–32.

184.

Sun

Fan

. Overexpression of Glypican 5 (GPC5) inhibits prostate cancer cell proliferation and invasion via suppressing Sp1-mediated EMT and activation of Wnt/beta-catenin signaling. Oncol Res. 2018 May 7;26(4):565–72.

185.

Veugelers

De Cat

Ceulemans

Bruystens

Coomans

Durr

Vermeesch

Marynen

David

. Glypican-6, a new member of the glypican family of cell surface heparan sulfate proteoglycans. J Biol Chem. 1999 Sep 17;274(38):26968–77.

186.

Dinccelik-Aslan

Gumus-Akay

Elhan

Unal

Tukun

. Diagnostic and prognostic significance of glypican 5 and glypican 6 gene expression levels in gastric adenocarcinoma. Mol Clin Oncol. 2015 May;3(3):584–90.

187.

Karapetsas

Giannakakis

Dangaj

Lanitis

Kynigopoulos

Lambropoulou

Tanyi

Galanis

Kakolyris

Trypsianis

Coukos

Sandaltzopoulos

. Overexpression of GPC6 and TMEM132D in early stage ovarian cancer correlates with CD8+ T-lymphocyte infiltration and increased patient survival. Biomed Res Int. 2015;2015:712438.

188.

Loftus

Baxter

Cronin

Fufa

Program

NCS

Pavan

. Hypoxia-induced HIF1alpha targets in melanocytes reveal a molecular profile associated with poor melanoma prognosis. Pigment Cell Melanoma Res. 2017 May;30(3):339–52.

189.

Shats

Krahn

Flake

Umbach

. Glypican 6 is a putative biomarker for metastatic progression of cutaneous melanoma. PLoS ONE. 2019;14(6):e0218067.

190.

Januchowski

Zawierucha

Rucinski

Nowicki

Zabel

. Extracellular matrix proteins expression profiling in chemoresistant variants of the A2780 ovarian cancer cell line. Biomed Res Int. 2014;2014:365867.

191.

Lau

Wong

Fan

Mak

Wong

Lam

Pang

Choy

. Allelic imbalance at 13q31 is associated with reduced GPC6 in Chinese with sporadic retinoblastoma. Br J Ophthalmol. 2010 Mar;94(3):357–62.

192.

Kumar

White

MacKenzie

Clegg

Lee

Dumpit

Coleman

Salipante

Rieder

Nickerson

Corey

Lange

Morrissey

Vessella

Nelson

Shendure

. Exome sequencing identifies a spectrum of mutation frequencies in advanced and lethal prostate cancers. Proc Natl Acad Sci U S A. 2011 Oct 11;108(41):17087–92.

193.

Amankwah

Lin

Tyrer

Lawrenson

Dennis

Chornokur

Aben

Anton-Culver

Antonenkova

Bruinsma

Bandera

Bean

Beckmann

Bisogna

Bjorge

Bogdanova

Brinton

Brooks-Wilson

Bunker

Butzow

Campbell

Carty

Chen

Chang-Claude

Cook

Cramer

Cunningham

Cybulski

Dansonka-Mieszkowska

Bois A

Despierre

Dicks

Doherty

Dork

Durst

Easton

Eccles

Edwards

Ekici

Fasching

Fridley

Gao

Gentry-Maharaj

Giles

Glasspool

Goodman

Gronwald

Harrington

Harter

Hasmad

Hein

Heitz

Hildebrandt

Hillemanns

Hogdall

Hosono

Iversen

Jakubowska

Jensen

Karlan

Jim

Kellar

Kiemeney

Krakstad

Kjaer

Kupryjanczyk

Lambrechts

Lee

Lele

Leminen

Lester

Levine

Liang

Lim

Lissowska

Lubinski

Lundvall

Massuger

Matsuo

McGuire

McLaughlin

McNeish

Menon

Milne

Modugno

Moysich

Ness

Nevanlinna

Eilber

Odunsi

Olson

Orlow

Orsulic

Weber

Paul

Pearce

Pejovic

Pelttari

Permuth-Wey

Pike

Poole

Risch

Rosen

Rossing

Rothstein

Rudolph

Runnebaum

Rzepecka

Salvesen

Schernhammer

Schwaab

Shu

Shvetsov

Siddiqui

Sieh

Song

Southey

Spiewankiewicz

Sucheston-Campbell

Teo

Terry

Thompson

Thomsen

Tangen

Tworoger

van Altena

Vierkant

Vergote

Walsh

Wang-Gohrke

Wentzensen

Whittemore

Wicklund

Wilkens

Woo

Yang

Zheng

Ziogas

Kelemen

Berchuck

Georgia Chenevix-Trench on behalf of the Amg Schildkraut JM Ramus

Goode

Monteiro

Gayther

Narod

Pharoah

Sellers

Phelan

. Epithelial-mesenchymal transition (EMT) gene variants and epithelial ovarian cancer (EOC) risk. Genet Epidemiol. 2015 Dec;39(8):689–97.

194.

Lebensohn

Dubey

Neitzel

Tacchelly-Benites

Yang

Marceau

Davis

Patel

Bahrami-Nejad

Travaglini

Ahmed

Lee

Carette

Rohatgi

. Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling. Elife. 2016 Dec 20;5:e21459.

195.

Yiu

Kaunisto

Chin

Toker

. NFAT promotes carcinoma invasive migration through glypican-6. Biochem J. 2011 Nov 15;440(1):157–66.