Targeting GPC3: Strategies for Therapeutic Intervention in Cancer

The Structure of GPC3

GPC3, or Glypican-3, is an oncofetal glycoprotein tethered to the cell membrane via a glycophosphatidylinositol (GPI) anchor[1-2]. Its core protein comprises 580 amino acids and weighs in at 70 kDa. Two heparan sulfate (HS) side chains are attached near the C-terminal section. A furin-mediated cleavage event at the Arg358-Cys359 bond processes the single-chain GPC3 into its mature form, composed of a 40-kDa N-terminal subunit and a 30-kDa C-terminal subunit connected by disulfide bonds[3]. Various GPC3 forms have been observed in the culture supernatant of GPC3-expressing cells and in serum, suggesting potential proteolytic cleavages of its extracellular segment[4].

The GPC3 gene is situated on the X chromosome (Xq26.2) and is recognized as a pivotal regulator of cellular proliferation in embryonic mesodermal tissues. Deletion of the GPC3 gene leads to the development of gigantism/overgrowth syndrome known as Simpson–Golabi–Behmel syndrome (SGBS)[5-6]. Mechanistically, GPC3 likely participates in the regulation of signaling pathways such as Wnt, hedgehog, bone morphogenic protein, and FGF. Through these pathways, it exerts control over cell growth and apoptosis in specific cell types during development[7-8]. GPC3 exhibits widespread expression in embryonic tissues like the placenta, as well as in the liver, lungs, and kidneys. However, in most adult organs, GPC3 is scarcely detectable. This biological downregulation in adult tissues may stem from DNA methylation within the GPC3 promoter region[9-10].

Function of GPC3

GPC3 exhibits a versatile nature in its ability to regulate cell growth, with its effects varying depending on the cell type involved.

In mesodermal embryonic tissues, GPC3 takes on a predominantly negative role. The deletion of the GPC3 gene has been linked to the pathogenesis of Simpson-Golabi-Behmel overgrowth syndrome[11]. Intriguingly, research has unveiled that GPC3 can interact with IGF2, effectively dampening IGF2-mediated growth in vivo. This evidence points toward GPC3's capacity to negatively regulate embryonic and fetal development. Additionally, GPC3 emerges as a negative transcriptional regulator and tumor suppressor, actively curbing the growth of breast, ovary, and lung cancer cells[12-13].

Conversely, in hepatocellular carcinoma (HCC), GPC3 assumes a pro-growth role. It is highly expressed in 70–100% of HCC cases[18]. GPC3 engages with Wnt to facilitate Wnt/Frizzled binding, ultimately promoting HCC growth. When the expression of GPC3 is downregulated in cell cultures, Yap signaling is reduced. Remarkably, soluble GPC3 proteins (GPC3DGPI) function dominantly in a negative manner, competing with endogenous GPC3 to hinder HCC cell growth, possibly by neutralizing GPC3 binding molecules[14]. These studies underscore the proliferative impact of GPC3 in HCC.

GPC3 and Tumor Progression

GPC3 expression has been detected in a range of tumor types, including hepatocellular carcinoma (HCC), lung squamous cell carcinoma (SqCC), gastric carcinoma, ovarian carcinoma, melanomas, and pediatric embryonal tumors. Notably, its expression is particularly elevated in HCC[16-17].

In HCC cells, GPC3 seems to play a significant role in the Wnt/β-catenin signaling pathway, bolstering cell proliferation. The core protein of GPC3 interacts with the Wnt receptor Frizzled (FZD). Recent research has unveiled that the GPC3 core protein acts as a co-receptor for Wnt, promoting Wnt/β-catenin signaling in HCC cells[18]. Upregulated GPC3 accelerates lung SqCC cell progression through a Wnt/β-catenin-dependent mechanism[19]. Furthermore, an enzyme called sulfatase 2 (SULF2) is upregulated in HCC cells, leading to increased release of heparin-binding growth factors, like FGF and HGF, which are attached to the HS sidechains of GPC3. This, in turn, activates signaling pathways mediated by their specific receptors[20-21]. Since Wnt can also attach to HS, SULF2 may enhance Wnt signaling as well. Conversely, soluble GPC3 has been found to suppress cancer cell proliferation[22]. The cleavage of the GPI anchor by sheddases like phospholipase D is thought to release cell surface GPC3. While notum was initially considered a sheddase of GPC3, it has been established that notum acts as a deacetylase, cleaving the palmitoleate moiety of Wnt attached to the HS chain, not the GPI anchor of GPC3, and inhibiting the binding of Wnt to FZD[23]. This suggests that GPC3 may serve as a crucial molecule in cancer cell biology, with cell surface GPI-anchored GPC3 potentially acting as a reservoir and cofactor for paracrine or autocrine growth factors to efficiently transmit their outside-in signaling. Conversely, shedding of GPC3 from the cell surface likely disrupts these signaling pathways. Hence, further studies are necessary to comprehend the molecular mechanisms and regulation of GPC3 shedding from cancer cell surfaces.

Recently, microRNAs (miRNAs) and long noncoding RNAs (lncRNA) have emerged as key regulators of gene expression in cancer cells[24]. Several miRNAs and lncRNAs have been identified as promoters or suppressors of the GPC3/Wnt/β-catenin axis in HCC[25]. Further investigations are warranted to gain deeper insights into the role of miRNAs, lncRNAs, and related axes that are critical in governing GPC3 expression.

Signaling Pathway of GPC3

In the progression of hepatocellular carcinoma (HCC), the activation of canonical Wnt signaling is a prevalent molecular event[26]. In fact, roughly 95% of HCC cases display some form of Wnt/β-catenin deregulation. This Wnt signaling pathway goes awry in various human diseases, including cancers and metabolic disorders.

Humans boast a total of 19 Wnt proteins, secreted through autocrine and paracrine systems. Canonical Wnt signaling, which relies on β-catenin, is initiated when Wnt molecules bind to two coreceptors: frizzled (FZD), a seven-pass transmembrane G protein-coupled receptor (GPCR), and low-density lipoprotein receptor-related protein 5/6 (LRP5/6), a single-pass transmembrane receptor. There are a total of 10 human FZDs[27]. Wnt ligands latch onto FZD's extracellular cysteine-rich domain, which houses the Wnt binding domain. This binding triggers the assembly of the FZD-LRP5/6 receptor complex[28]. Subsequent conformational changes in FZD and LRP5/6, along with the phosphorylation of glycogen synthase kinase 3 and casein kinase 1, facilitate the recruitment of Axin, a critical component of the destruction complex. This, in turn, leads to the recruitment of DVL, a cytoplasmic protein, binding to the C-terminal tail of FZD. Consequently, the destruction complex, which includes DVL, Axin, and other binding partners, becomes stabilized[29]. Axin prevents the degradation of β-catenin, allowing it to accumulate in the cytoplasm. From there, β-catenin translocates to the nucleus, where it drives the transcription of genes responsible for cell proliferation and survival[30]. It's worth noting that Wnt signaling can also function through a β-catenin-independent pathway, known as the non-canonical or alternative pathway[31].

Given the crucial role of Wnt signaling in functions like hepatobiliary processes, cell differentiation, and repair, any dysregulation in this pathway can lead to HCC, hepatoblastoma, cholangiocarcinoma, or other liver diseases.

GPC3 Protein

Recombinant Human GPC3 Protein (C-6His)

Click here for more GPC3

Synonym : Glypican-3; GTR2-2; Intestinal protein OCI-5; MXR7; GPC3; OCI5

References:

[1] Filmus J, Capurro M. Glypican-3: a marker and a therapeutic target in hepatocellular carcinoma. FEBS J. 2013 May;280(10):2471-6. doi: 10.1111/febs.12126. Epub 2013 Jan 31. PMID: 23305321.

[2] Filmus J, Capurro M, Rast J. Glypicans. Genome Biol. 2008;9(5):224. doi: 10.1186/gb-2008-9-5-224. Epub 2008 May 22. PMID: 18505598; PMCID: PMC2441458.

[3] De Cat B, Muyldermans SY, Coomans C, Degeest G, Vanderschueren B, Creemers J, Biemar F, Peers B, David G. Processing by proprotein convertases is required for glypican-3 modulation of cell survival, Wnt signaling, and gastrulation movements. J Cell Biol. 2003 Nov 10;163(3):625-35. doi: 10.1083/jcb.200302152. PMID: 14610063; PMCID: PMC2173654.

[4] Hippo Y, Watanabe K, Watanabe A, Midorikawa Y, Yamamoto S, Ihara S, Tokita S, Iwanari H, Ito Y, Nakano K, Nezu J, Tsunoda H, Yoshino T, Ohizumi I, Tsuchiya M, Ohnishi S, Makuuchi M, Hamakubo T, Kodama T, Aburatani H. Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma. Cancer Res. 2004 Apr 1;64(7):2418-23. doi: 10.1158/0008-5472.can-03-2191. PMID: 15059894.

[5] Golabi M, Rosen L. A new X-linked mental retardation-overgrowth syndrome. Am J Med Genet. 1984 Jan;17(1):345-58. doi: 10.1002/ajmg.1320170128. PMID: 6538755.

[6] Behmel A, Plöchl E, Rosenkranz W. A new X-linked dysplasia gigantism syndrome: identical with the Simpson dysplasia syndrome? Hum Genet. 1984;67(4):409-13. doi: 10.1007/BF00291401. PMID: 6490008.

[7] Iglesias BV, Centeno G, Pascuccelli H, Ward F, Peters MG, Filmus J, Puricelli L, de Kier Joffé EB. Expression pattern of glypican-3 (GPC3) during human embryonic and fetal development. Histol Histopathol. 2008 Nov;23(11):1333-40. doi: 10.14670/HH-23.1333. PMID: 18785116.

[8] Paine-Saunders S, Viviano BL, Zupicich J, Skarnes WC, Saunders S. glypican-3 controls cellular responses to Bmp4 in limb patterning and skeletal development. Dev Biol. 2000 Sep 1;225(1):179-87. doi: 10.1006/dbio.2000.9831. PMID: 10964473.

[9] Huber R, Hansen RS, Strazzullo M, Pengue G, Mazzarella R, D'Urso M, Schlessinger D, Pilia G, Gartler SM, D'Esposito M. DNA methylation in transcriptional repression of two differentially expressed X-linked genes, GPC3 and SYBL1. Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):616-21. doi: 10.1073/pnas.96.2.616. PMID: 9892682; PMCID: PMC15185.

[10] Boily G, Saikali Z, Sinnett D. Methylation analysis of the glypican 3 gene in embryonal tumours. Br J Cancer. 2004 Apr 19;90(8):1606-11. doi: 10.1038/sj.bjc.6601716. PMID: 15083193; PMCID: PMC2409702.

[11] Pilia G, Hughes-Benzie RM, MacKenzie A, Baybayan P, Chen EY, Huber R, Neri G, Cao A, Forabosco A, Schlessinger D. Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-Behmel overgrowth syndrome. Nat Genet. 1996 Mar;12(3):241-7. doi: 10.1038/ng0396-241. PMID: 8589713.

[12] Xiang YY, Ladeda V, Filmus J. Glypican-3 expression is silenced in human breast cancer. Oncogene. 2001 Nov 1;20(50):7408-12. doi: 10.1038/sj.onc.1204925. PMID: 11704870.

[13] Kim H, Xu GL, Borczuk AC, Busch S, Filmus J, Capurro M, Brody JS, Lange J, D'Armiento JM, Rothman PB, Powell CA. The heparan sulfate proteoglycan GPC3 is a potential lung tumor suppressor. Am J Respir Cell Mol Biol. 2003 Dec;29(6):694-701. doi: 10.1165/rcmb.2003-0061OC. Epub 2003 Jun 19. PMID: 12816733.

[14] Zittermann SI, Capurro MI, Shi W, Filmus J. Soluble glypican 3 inhibits the growth of hepatocellular carcinoma in vitro and in vivo. Int J Cancer. 2010 Mar 15;126(6):1291-301. doi: 10.1002/ijc.24941. PMID: 19816934.

[15] Shih TC, Wang L, Wang HC, Wan YY. Glypican-3: A molecular marker for the detection and treatment of hepatocellular carcinoma☆. Liver Res. 2020 Dec;4(4):168-172. doi: 10.1016/j.livres.2020.11.003. Epub 2020 Nov 11. PMID: 33384879; PMCID: PMC7771890.

[16] Lin Q, Xiong LW, Pan XF, Gen JF, Bao GL, Sha HF, Feng JX, Ji CY, Chen M. Expression of GPC3 protein and its significance in lung squamous cell carcinoma. Med Oncol. 2012 Jun;29(2):663-9. doi: 10.1007/s12032-011-9973-1. Epub 2011 May 10. PMID: 21556932.

[17] Maeda D, Ota S, Takazawa Y, Aburatani H, Nakagawa S, Yano T, Taketani Y, Kodama T, Fukayama M. Glypican-3 expression in clear cell adenocarcinoma of the ovary. Mod Pathol. 2009 Jun;22(6):824-32. doi: 10.1038/modpathol.2009.40. Epub 2009 Mar 27. PMID: 19329941.

[18] Li N, Wei L, Liu X, Bai H, Ye Y, Li D, Li N, Baxa U, Wang Q, Lv L, Chen Y, Feng M, Lee B, Gao W, Ho M. 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):1231-1245. doi: 10.1002/hep.30646. Epub 2019 May 24. PMID: 30963603; PMCID: PMC6783318.

[19] Wang D, Gao Y, Zhang Y, Wang L, Chen G. Glypican-3 promotes cell proliferation and tumorigenesis through up-regulation of β-catenin expression in lung squamous cell carcinoma. Biosci Rep. 2019 Jun 25;39(6):BSR20181147. doi: 10.1042/BSR20181147. PMID: 31160489; PMCID: PMC6591568.

[20] Wu Y, Liu H, Weng H, Zhang X, Li P, Fan CL, Li B, Dong PL, Li L, Dooley S, Ding HG. Glypican-3 promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells through ERK signaling pathway. Int J Oncol. 2015 Mar;46(3):1275-85. doi: 10.3892/ijo.2015.2827. Epub 2015 Jan 9. PMID: 25572615.

[21] Lv G, Tan Y, Lv H, Fang T, Wang C, Li T, Yu Y, Hu C, Wen W, Wang H, Yang W. MXR7 facilitates liver cancer metastasis via epithelial-mesenchymal transition. Sci China Life Sci. 2017 Nov;60(11):1203-1213. doi: 10.1007/s11427-016-9042-y. Epub 2017 Aug 11. PMID: 28812296.

[22] Saad, A.; Liet, B.; Joucla, G.; Santarelli, X.; Charpentier, J.; Claverol, S.; Grosset, C.F.; Trézéguet, V. Role of Glycanation and Convertase Maturation of Soluble Glypican-3 in Inhibiting Proliferation of Hepatocellular Carcinoma Cells. Biochemistry 2018, 57, 1201–1211.

[23] Kakugawa S, Langton PF, Zebisch M, Howell S, Chang TH, Liu Y, Feizi T, Bineva G, O'Reilly N, Snijders AP, Jones EY, Vincent JP. Notum deacylates Wnt proteins to suppress signalling activity. Nature. 2015 Mar 12;519(7542):187-192. doi: 10.1038/nature14259. Epub 2015 Feb 25. PMID: 25731175; PMCID: PMC4376489.

[24] Montani F, Bianchi F. Circulating Cancer Biomarkers: The Macro-revolution of the Micro-RNA. EBioMedicine. 2016 Feb 28;5:4-6. doi: 10.1016/j.ebiom.2016.02.038. PMID: 27077096; PMCID: PMC4816844.

[25] Zhu XT, Yuan JH, Zhu TT, Li YY, Cheng XY. Long noncoding RNA glypican 3 (GPC3) antisense transcript 1 promotes hepatocellular carcinoma progression via epigenetically activating GPC3. FEBS J. 2016 Oct;283(20):3739-3754. doi: 10.1111/febs.13839. Epub 2016 Sep 21. PMID: 27573079.

[26] Pez F, Lopez A, Kim M, Wands JR, Caron de Fromentel C, Merle P. Wnt signaling and hepatocarcinogenesis: molecular targets for the development of innovative anticancer drugs. J Hepatol. 2013 Nov;59(5):1107-17. doi: 10.1016/j.jhep.2013.07.001. Epub 2013 Jul 5. PMID: 23835194.

[27] Nusse R, Clevers H. Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell. 2017 Jun 1;169(6):985-999. doi: 10.1016/j.cell.2017.05.016. PMID: 28575679.

[28] Cong F, Schweizer L, Varmus H. Wnt signals across the plasma membrane to activate the beta-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP. Development. 2004 Oct;131(20):5103-15. doi: 10.1242/dev.01318. PMID: 15459103.

[29] Gao C, Chen YG. Dishevelled: The hub of Wnt signaling. Cell Signal. 2010 May;22(5):717-27. doi: 10.1016/j.cellsig.2009.11.021. Epub 2009 Dec 13. PMID: 20006983.

[30] Vlad A, Röhrs S, Klein-Hitpass L, Müller O. The first five years of the Wnt targetome. Cell Signal. 2008 May;20(5):795-802. doi: 10.1016/j.cellsig.2007.10.031. Epub 2007 Nov 17. PMID: 18160255.

[31] van Amerongen R. Alternative Wnt pathways and receptors. Cold Spring Harb Perspect Biol. 2012 Oct 1;4(10):a007914. doi: 10.1101/cshperspect.a007914. PMID: 22935904; PMCID: PMC3475174.

[32] Kolluri A, Ho M. The Role of Glypican-3 in Regulating Wnt, YAP, and Hedgehog in Liver Cancer. Front Oncol. 2019 Aug 2;9:708. doi: 10.3389/fonc.2019.00708. PMID: 31428581; PMCID: PMC6688162.