Abstract
Purpose
We have shown that GPC3 overexpression in breast cancer cells inhibits in vivo tumor progression, by acting as a metastatic suppressor. GPC3-overexpressing cells are less clonogenic, viable and motile, while their homotypic adhesion is increased. We have presented evidences indicating that GPC3 inhibits canonical Wnt and Akt pathways, while non-canonical Wnt and p38MAPK cascades are activated. In this study, we aimed to investigate whether GPC3-induced Wnt signaling inhibition modulates breast cancer cell properties as well as to describe the interactions among pathways modulated by GPC3.
Methods
Fluorescence microscopy, qRT-PCR microarray, gene reporter assay and Western blotting were performed to determine gene expression levels, signaling pathway activities and molecule localization. Lithium was employed to activate canonical Wnt pathway and treated LM3-GPC3 cell viability, migration, cytoskeleton organization and homotypic adhesion were assessed using MTS, wound healing, phalloidin staining and suspension growth assays, respectively.
Results
We provide new data demonstrating that GPC3 blocks—also at a transcriptional level—both autocrine and paracrine canonical Wnt activities, and that this inhibition is required for GPC3 to modulate migration and homotypic adhesion. Our results indicate that GPC3 is secreted into the extracellular media, suggesting that secreted GPC3 competes with Wnt factors or interacts with them and thus prevents Wnt binding to Fz receptors. We also describe the complex network of interactions among GPC3-modulated signaling pathways.
Conclusion
GPC3 is operating through an intricate molecular signaling network. From the balance of these interactions, the inhibition of breast metastatic spread induced by GPC3 emerges.
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References
Akiyama T, Kamimura K, Firkus C, Takeo S, Shimmi O, Nakato H (2008) Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface. Dev Biol 313:408–419
Amin N, Vincan E (2012) The Wnt signaling pathways and cell adhesion. Front Biosci (Landmark Ed) 17:784–804
Baeg GH, Perrimon N (2000) Functional binding of secreted molecules to heparan sulfate proteoglycans in Drosophila. Curr Opin Cell Biol 12:575–580
Baeg GH, Lin X, Khare N, Baumgartner S, Perrimon N (2001) Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128:87–94
Bafico A, Liu G, Goldin L, Harris V, Aaronson SA (2004) An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells. Cancer Cell 6:497–506. https://doi.org/10.1016/j.ccr.2004.09.032
Banerjee S, Gordon L, Donn TM, Berti C, Moens CB, Burden SJ, Granato M (2011) A novel role for MuSK and non-canonical Wnt signaling during segmental neural crest cell migration. Development 138:3287–3296. https://doi.org/10.1242/dev.067306
Benhaj K, Akcali KC, Ozturk M (2006) Redundant expression of canonical Wnt ligands in human breast cancer cell lines. Oncol Rep 15:701–707
Bikkavilli RK, Feigin ME, Malbon CC (2008a) G alpha o mediates WNT-JNK signaling through dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells. J Cell Sci 121:234–245. https://doi.org/10.1242/jcs.021964
Bikkavilli RK, Feigin ME, Malbon CC (2008b) p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta. J Cell Sci 121:3598–3607. https://doi.org/10.1242/jcs.032854
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Buchanan C et al (2010) Glypican-3 reexpression regulates apoptosis in murine adenocarcinoma mammary cells modulating PI3K/Akt and p38MAPK signaling pathways. Breast Cancer Res Treat 119:559–574. https://doi.org/10.1007/s10549-009-0362-9
Buchanan C, Lago Huvelle MA, Peters MG (2011) Metastasis suppressors: basic and translational advances. Curr Pharm Biotechnol 12:1948–1960
Cai K, Jiang L, Wang J, Zhang H, Wang X, Cheng D, Dou J (2014) Downregulation of beta-catenin decreases the tumorigenicity, but promotes epithelial-mesenchymal transition in breast cancer cells. J Cancer Res Ther 10:1063–1070. https://doi.org/10.4103/0973-1482.139378
Cano-Gauci DF et al (1999) Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson–Golabi–Behmel syndrome. J Cell Biol 146:255–264
Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, Filmus J (2003) Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology 125:89–97
Capurro MI, Xiang YY, Lobe C, Filmus J (2005) Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical. Wnt Signaling Cancer Res 65:6245–6254
Capurro MI, Xu P, Shi W, Li F, Jia A, Filmus J (2008) Glypican-3 inhibits Hedgehog signaling during development by competing with patched for Hedgehog binding. Dev Cell 14:700–711. https://doi.org/10.1016/j.devcel.2008.03.006
Capurro M, Martin T, Shi W, Filmus J (2014) Glypican-3 binds to Frizzled and plays a direct role in the stimulation of canonical Wnt signaling. J Cell Sci 127:1565–1575. https://doi.org/10.1242/jcs.140871
Castillo L et al (2015) Expression of Glypican-3 (GPC3) in malignant and non-malignant human breast tissues. Open Cancer J 8:12–23. https://doi.org/10.2174/1874079001508010012
Castillo LF et al (2016) Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells. Oncotarget. https://doi.org/10.18632/oncotarget.11107
Chang J et al (2007) Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK. J Biol Chem 282:30938–30948. https://doi.org/10.1074/jbc.M702391200
Desbordes SC, Sanson B (2003) The glypican Dally-like is required for Hedgehog signalling in the embryonic epidermis of Drosophila. Development 130:6245–6255
Dihlmann S, Klein S, Doeberitz Mv M (2003) Reduction of beta-catenin/T-cell transcription factor signaling by aspirin and indomethacin is caused by an increased stabilization of phosphorylated beta-catenin. Mol Cancer Ther 2:509–516
Dwivedi PP, Grose RH, Filmus J, Hii CS, Xian CJ, Anderson PJ, Powell BC (2013) Regulation of bone morphogenetic protein signalling and cranial osteogenesis by Gpc1 and Gpc3. Bone 55:367–376. https://doi.org/10.1016/j.bone.2013.04.013
Fang D et al (2007) Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity. J Biol Chem 282:11221–11229. https://doi.org/10.1074/jbc.M611871200
Feng M, Kim H, Phung Y, Ho M (2011) Recombinant soluble glypican 3 protein inhibits the growth of hepatocellular carcinoma in vitro. Int J Cancer 128:2246–2247. https://doi.org/10.1002/ijc.25549
Filmus J, Shi W, Wong ZM, Wong MJ (1995) Identification of a new membrane-bound heparan sulphate proteoglycan. Biochem J 311(Pt 2):561–565
Force T, Woulfe K, Koch WJ, Kerkela R (2007) Molecular scaffolds regulate bidirectional crosstalk between Wnt and classical seven-transmembrane-domain receptor signaling pathways. Sci STKE Signal Transduct Knowl Environ 2007:pe41. https://doi.org/10.1126/stke.3972007pe41
Gottardi CJ, Gumbiner BM (2004) Distinct molecular forms of beta-catenin are targeted to adhesive or transcriptional complexes. J Cell Biol 167:339–349. https://doi.org/10.1083/jcb.200402153
Gutierrez J, Brandan E (2010) A novel mechanism of sequestering fibroblast growth factor 2 by glypican in lipid rafts allowing skeletal muscle differentiation. Mol Cell Biol 30:1634–1649. https://doi.org/10.1128/MCB.01164-09
Han C, Yan D, Belenkaya TY, Lin X (2005) Drosophila glypicans Dally and Dally-like shape the extracellular Wingless morphogen gradient in the wing disk. Development 132:667–679
Han S et al (2016) Identification of Glypican-3 as a potential metastasis suppressor gene in gastric cancer. Oncotarget 7:44406–44416. https://doi.org/10.18632/oncotarget.9763
Hankey W, Frankel WL, Groden J (2018) Functions of the APC tumor suppressor protein dependent and independent of canonical WNT signaling: implications for therapeutic targeting. Cancer Metastasis Rev 37:159–172. https://doi.org/10.1007/s10555-017-9725-6
Hartsock A, Nelson WJ (2008) Adherens and tight junctions: structure, function and connections to the actin cytoskeleton. Biochimica et Biophysica Acta 1778:660–669. https://doi.org/10.1016/j.bbamem.2007.07.012
Hippo Y et al (2004) Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma. Cancer Res 64:2418–2423
Iglesias BV et al (2008) Expression pattern of glypican-3 (GPC3) during human embryonic and fetal development. Histol Histopathol 23:1333–1340
Ishitani T et al (1999) The TAK1-NLK-MAPK-related pathway antagonizes signalling between beta-catenin and transcription factor. TCF Nat 399:798–802. https://doi.org/10.1038/21674
Ishitani T et al (2003) The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling. Mol Cell Biol 23:131–139
Khare N, Baumgartner S (2000) Dally-like protein, a new Drosophila glypican with expression overlapping with wingless. Mech Dev 99:199–202
Kim AH, Khursigara G, Sun X, Franke TF, Chao MV (2001) Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol Cell Biol 21:893–901. https://doi.org/10.1128/MCB.21.3.893-901.2001
Kim H et al (2003) The heparan sulfate proteoglycan GPC3 is a potential lung tumor suppressor. Am J Respir Cell Mol Biol 29:694–701. https://doi.org/10.1165/rcmb.2003-0061OC
Kim JH et al (2010) Beta-catenin regulates melanocyte dendricity through the modulation of PKCzeta and PKCdelta. Pigment Cell Melanoma Res 23:385–393. https://doi.org/10.1111/j.1755-148X.2010.00695.x
Klein PS, Melton DA (1996) A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci USA 93:8455–8459
Kreuger J, Perez L, Giraldez AJ, Cohen SM (2004) Opposing activities of Dally-like glypican at high and low levels of Wingless morphogen activity. Dev Cell 7:503–512
Krishnamurthy N, Kurzrock R (2018) Targeting the Wnt/beta-catenin pathway in cancer: update on effectors and inhibitors. Cancer Treat Rev 62:50–60. https://doi.org/10.1016/j.ctrv.2017.11.002
Li G et al (2018) Frizzled7 promotes epithelial-to-mesenchymal transition and stemness via activating canonical Wnt/beta-catenin pathway in gastric cancer. Int J Biol Sci 14:280–293. https://doi.org/10.7150/ijbs.23756
Lin H, Huber R, Schlessinger D, Morin PJ (1999) Frequent silencing of the GPC3 gene in ovarian cancer cell lines. Cancer Res 59:807–810
Lin CL, Wang JY, Huang YT, Kuo YH, Surendran K, Wang FS (2006) Wnt/beta-catenin signaling modulates survival of high glucose-stressed mesangial cells. J Am Soc Nephrol 17:2812–2820. https://doi.org/10.1681/ASN.2005121355
Ma L, Wang HY (2007) Mitogen-activated protein kinase p38 regulates the Wnt/cyclic GMP/Ca2+ non-canonical pathway. J Biol Chem 282:28980–28990. https://doi.org/10.1074/jbc.M702840200
Ma J et al (2010) IGF-1 mediates PTEN suppression and enhances cell invasion and proliferation via activation of the IGF-1/PI3K/Akt signaling pathway in pancreatic cancer cells. J Surg Res 160:90–101. https://doi.org/10.1016/j.jss.2008.08.016
Maeda D, Ota S, Takazawa Y, Aburatani H, Nakagawa S, Yano T, Taketani Y, Kodama T, Fukayama M (2009) Glypican-3 expression in clear cell adenocarcinoma of the ovary. Mod Pathol 22:824–832. https://doi.org/10.1038/modpathol.2009.40
Maguire PB, Donlon T, Parsons M, Wynne K, Dillon E, Ni Ainle F, Szklanna PB (2018) Proteomic analysis reveals a strong association of beta-catenin with cadherin adherens junctions in resting. Hum Platelets Proteom 18:e1700419. https://doi.org/10.1002/pmic.201700419
Mitsiades CS, Mitsiades N, Koutsilieris M (2004) The Akt pathway: molecular targets for anti-cancer drug development. Curr Cancer Drug Targets 4:235–256
Mizushima S, Nagata S (1990) pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res 18:5322
Montalbano M, Rastellini C, McGuire JT, Prajapati J, Shirafkan A, Vento R, Cicalese L (2018) Role of Glypican-3 in the growth, migration and invasion of primary hepatocytes isolated from patients with hepatocellular carcinoma. Cell Oncol (Dordr) 41:169–184. https://doi.org/10.1007/s13402-017-0364-2
Murthy SS et al (2000) Expression of GPC3, an X-linked recessive overgrowth gene is silenced in malignant mesothelioma. Oncogene 19:410–416. https://doi.org/10.1038/sj.onc.1203322
Pellegrini M et al (1998) Gpc3 expression correlates with the phenotype of the Simpson–Golabi–Behmel syndrome. Dev Dyn 213:431–439
Peters MG, Farias E, Colombo L, Filmus J, Puricelli L, Bal de Kier Joffe E (2003) Inhibition of invasion and metastasis by glypican-3 in a syngeneic breast cancer model. Breast Cancer Res Treat 80:221–232
Pilia G et al (1996) Mutations in GPC3, a glypican gene, cause the Simpson–Golabi–Behmel overgrowth syndrome. Nat Genet 12:241–247
Princivalle M, de Agostini A (2002) Developmental roles of heparan sulfate proteoglycans: a comparative review in Drosophila, mouse and human. Int J Dev Biol 46:267–278
Roarty K, Pfefferle AD, Creighton CJ, Perou CM, Rosen JM (2017) Ror2-mediated alternative Wnt signaling regulates cell fate and adhesion during mammary tumor progression. Oncogene 36:5958–5968. https://doi.org/10.1038/onc.2017.206
Rosso SB, Sussman D, Wynshaw-Boris A, Salinas PC (2005) Wnt signaling through Dishevelled, Rac and JNK regulates dendritic development. Nat Neurosci 8:34–42. https://doi.org/10.1038/nn1374
Saad K, Theis S, Otto A, Luke G, Patel K (2017) Detailed expression profile of the six Glypicans and their modifying enzyme, Notum during chick limb feather development. Gene 610:71–79. https://doi.org/10.1016/j.gene.2017.02.012
Sakane H, Yamamoto H, Matsumoto S, Sato A, Kikuchi A (2012) Localization of glypican-4 in different membrane microdomains is involved in the regulation of Wnt signaling. J Cell Sci 125:449–460. https://doi.org/10.1242/jcs.091876
Sharma M, Castro-Piedras I, Simmons GE Jr, Pruitt K (2018) Dishevelled: a masterful conductor of complex Wnt signals. Cell Signal 47:52–64. https://doi.org/10.1016/j.cellsig.2018.03.004
Song HH, Shi W, Xiang YY, Filmus J (2005) The loss of glypican-3 induces alterations in Wnt signaling. J Biol Chem 280:2116–2125
Stigliano I, Puricelli L, Filmus J, Sogayar MC, Bal de Kier Joffe E, Peters MG (2009) Glypican-3 regulates migration, adhesion and actin cytoskeleton organization in mammary tumor cells through Wnt signaling modulation. Breast Cancer Res Treat 114:251–262. https://doi.org/10.1007/s10549-008-0009-2
Sugimura J et al (2004) Gene expression profiling of early- and late-relapse nonseminomatous germ cell tumor and primitive neuroectodermal tumor of the testis. Clin Cancer Res Off J Am Assoc Cancer Res 10:2368–2378
Sun TQ, Lu B, Feng JJ, Reinhard C, Jan YN, Fantl WJ, Williams LT (2001) PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling. Nat Cell Biol 3:628–636. https://doi.org/10.1038/35083016
Tangkijvanich P, Chanmee T, Komtong S, Mahachai V, Wisedopas N, Pothacharoen P, Kongtawelert P (2010) Diagnostic role of serum glypican-3 in differentiating hepatocellular carcinoma from non-malignant chronic liver disease and other liver cancers. J Gastroenterol Hepatol 25:129–137. https://doi.org/10.1111/j.1440-1746.2009.05988.x
Toretsky JA et al (2001) Glypican-3 expression in Wilms tumor and hepatoblastoma. J Pediatr Hematol Oncol 23:496–499
Traister A, Shi W, Filmus J (2008) Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface. Biochem J 410:503–511. https://doi.org/10.1042/BJ20070511
Tsuda M et al (1999) The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila. Nature 400:276–280
Urtreger A, Ladeda V, Puricelli L, Rivelli A, Vidal MC, Lustig ES, Bal de Kier Joffé E (1997) Modulation of fibronectin expression and proteolytic activity associated with the invasive and metastatic phenotype in two murine mammary cell lines. Int J Oncol 11:489–496
Valsechi MC et al (2014) GPC3 reduces cell proliferation in renal carcinoma cell lines. BMC Cancer 14:631. https://doi.org/10.1186/1471-2407-14-631
Veugelers M et al (1999) Glypican-6, a new member of the glypican family of cell surface heparan sulfate proteoglycans. J Biol Chem 274:26968–26977
Xu Y, Li N, Xiang R, Sun P (2014) Emerging roles of the p38 MAPK and PI3K/AKT/mTOR pathways in oncogene-induced senescence. Trends Biochem Sci 39:268–276. https://doi.org/10.1016/j.tibs.2014.04.004
Xu J, Lv W, Hu Y, Wang L, Wang Y, Cao J, Hu J (2017) Wnt3a expression is associated with epithelial-mesenchymal transition and impacts prognosis of lung adenocarcinoma. Patients J Cancer 8:2523–2531. https://doi.org/10.7150/jca.18560
Yan D, Wu Y, Feng Y, Lin SC, Lin X (2009) The core protein of glypican Dally-like determines its biphasic activity in wingless morphogen signaling. Dev Cell 17:470–481. https://doi.org/10.1016/j.devcel.2009.09.001
Zittermann SI, Capurro MI, Shi W, Filmus J (2010) Soluble glypican 3 inhibits the growth of hepatocellular carcinoma in vitro and in vivo. Int J Cancer 126:1291–1301. https://doi.org/10.1002/ijc.24941
Zuluaga S, Alvarez-Barrientos A, Gutierrez-Uzquiza A, Benito M, Nebreda AR, Porras A (2007) Negative regulation of Akt activity by p38alpha MAP kinase in cardiomyocytes involves membrane localization of PP2A through interaction with caveolin-1. Cell Signal 19:62–74. https://doi.org/10.1016/j.cellsig.2006.05.032
Acknowledgements
This work was supported by Argentine grants from FONCyT (PICT 2013-1337 Préstamo BID)—Ministry of Science and Technical, CONICET (PIP 2013-112200120100100CO) and Fundación Florencio Fiorini. We would like to thank Dr. Osvaldo Rey for assistance with immunofluorescence and Dr. Mariana Salatino for help with conditional medium concentration.
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432_2018_2751_MOESM1_ESM.tif
Fig. 1 GPC3 expression levels. A RT-PCR. Total RNA from LM3-GPC3 and LM3-vector cells was retrotranscribed and used as template in PCR reactions. Agarose gel representative images are shown. B WB. Total protein extracts from LM3-GPC3 and LM3-vector cells were analyzed by WB to determine GPC3 levels. The arrows indicate GPC3 full-length core protein and N-terminal subunit. Actin was employed as seeding control (TIF 1033 KB)
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Fernández, D., Guereño, M., Lago Huvelle, M.A. et al. Signaling network involved in the GPC3-induced inhibition of breast cancer progression: role of canonical Wnt pathway. J Cancer Res Clin Oncol 144, 2399–2418 (2018). https://doi.org/10.1007/s00432-018-2751-0
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DOI: https://doi.org/10.1007/s00432-018-2751-0