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Open Access 01-12-2021 | Research article

Cancer-associated mutations reveal a novel role for EpCAM as an inhibitor of cathepsin-L and tumor cell invasion

Authors: Narendra V. Sankpal, Taylor C. Brown, Timothy P. Fleming, John M. Herndon, Anusha A. Amaravati, Allison N. Loynd, William E. Gillanders

Published in: BMC Cancer | Issue 1/2021

Abstract

Background

EpCAM (Epithelial cell adhesion molecule) is often dysregulated in epithelial cancers. Prior studies implicate EpCAM in the regulation of oncogenic signaling pathways and epithelial-to-mesenchymal transition. It was recently demonstrated that EpCAM contains a thyroglobulin type-1 (TY-1) domain. Multiple proteins with TY-1 domains are known to inhibit cathepsin-L (CTSL), a cysteine protease that promotes tumor cell invasion and metastasis. Analysis of human cancer sequencing studies reveals that somatic EpCAM mutations are present in up to 5.1% of tested tumors.

Methods

The Catalogue of Somatic Mutations in Cancer (COSMIC) database was queried to tabulate the position and amino acid changes of cancer associated EpCAM mutations. To determine how EpCAM mutations affect cancer biology we studied C66Y, a damaging TY-1 domain mutation identified in liver cancer, as well as 13 other cancer-associated EpCAM mutations. In vitro and in vivo models were used to determine the effect of wild type (WT) and mutant EpCAM on CTSL activity and invasion. Immunoprecipitation and localization studies tested EpCAM and CTSL protein binding and determined compartmental expression patterns of EpCAM mutants.

Results

We demonstrate that WT EpCAM, but not C66Y EpCAM, inhibits CTSL activity in vitro, and the TY-1 domain of EpCAM is responsible for this inhibition. WT EpCAM, but not C66Y EpCAM, inhibits tumor cell invasion in vitro and lung metastases in vivo. In an extended panel of human cancer cell lines, EpCAM expression is inversely correlated with CTSL activity. Previous studies have demonstrated that EpCAM germline mutations can prevent EpCAM from being expressed at the cell surface. We demonstrate that C66Y and multiple other EpCAM cancer-associated mutations prevent surface expression of EpCAM. Cancer-associated mutations that prevent EpCAM cell surface expression abrogate the ability of EpCAM to inhibit CTSL activity and tumor cell invasion.

Conclusions

These studies reveal a novel role for EpCAM as a CTSL inhibitor, confirm the functional relevance of multiple cancer-associated EpCAM mutations, and suggest a therapeutic vulnerability in cancers harboring EpCAM mutations.

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Momburg F, Moldenhauer G, Hammerling GJ, Moller P. Immunohistochemical study of the expression of a Mr 34,000 human epithelium-specific surface glycoprotein in normal and malignant tissues. Cancer Res. 1987;47(11):2883–91.PubMed

Spizzo G, Went P, Dirnhofer S, Obrist P, Simon R, Spichtin H, et al. High Ep-CAM expression is associated with poor prognosis in node-positive breast cancer. Breast Cancer Res Treat. 2004;86(3):207–13. https://doi.org/10.1023/B:BREA.0000036787.59816.01.CrossRefPubMed

Went PT, Lugli A, Meier S, Bundi M, Mirlacher M, Sauter G, et al. Frequent EpCAM protein expression in human carcinomas. Hum Pathol. 2004;35(1):122–8. https://doi.org/10.1016/j.humpath.2003.08.026.CrossRefPubMed

Herlyn M, Steplewski Z, Herlyn D, Koprowski H. Colorectal carcinoma-specific antigen detection by means of monoclonal antibodies. Proc Natl Acad Sci U S A. 1979;76(3):1438–42. https://doi.org/10.1073/pnas.76.3.1438.CrossRefPubMedPubMedCentral

Sears HF, Atkinson B, Mattis J, Ernst C, Herlyn D, Steplewski Z, et al. Phase-I clinical trial of monoclonal antibody in treatment of gastrointestinal tumours. Lancet. 1982;1(8275):762–5. https://doi.org/10.1016/s0140-6736(82)91811-6.CrossRefPubMed

Riethmuller G, Holz E, Schlimok G, Schmiegel W, Raab R, Hoffken K, et al. Monoclonal antibody therapy for resected Dukes’ C colorectal cancer: seven-year outcome of a multicenter randomized trial. J Clin Oncol. 1998;16(5):1788–94. https://doi.org/10.1200/JCO.1998.16.5.1788.CrossRefPubMed

Linke R, Klein A, Seimetz D. Catumaxomab: clinical development and future directions. MAbs. 2010;2(2):129–36. https://doi.org/10.4161/mabs.2.2.11221.CrossRefPubMedPubMedCentral

Sankpal NV, Fleming TP, Sharma PK, Wiedner HJ, Gillanders WE. A double-negative feedback loop between EpCAM and ERK contributes to the regulation of epithelial-mesenchymal transition in cancer. Oncogene. 2017;36(26):3706–17. https://doi.org/10.1038/onc.2016.504.CrossRefPubMedPubMedCentral

Sankpal NV, Fleming TP, Gillanders WE. EpCAM modulates NF-kappaB signaling and interleukin-8 expression in breast cancer. Mol Cancer Res. 2013;11(4):418–26. https://doi.org/10.1158/1541-7786.MCR-12-0518.CrossRefPubMedPubMedCentral

10.

Sankpal NV, Mayfield JD, Willman MW, Fleming TP, Gillanders WE. Activator protein 1 (AP-1) contributes to EpCAM-dependent breast cancer invasion. Breast Cancer Res. 2011;13(6):R124. https://doi.org/10.1186/bcr3070.CrossRefPubMedPubMedCentral

11.

Gostner JM, Fong D, Wrulich OA, Lehne F, Zitt M, Hermann M, et al. Effects of EpCAM overexpression on human breast cancer cell lines. BMC Cancer. 2011;11(1):45. https://doi.org/10.1186/1471-2407-11-45.CrossRefPubMedPubMedCentral

12.

Yamashita T, Budhu A, Forgues M, Wang XW. Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma. Cancer Res. 2007;67(22):10831–9. https://doi.org/10.1158/0008-5472.CAN-07-0908.CrossRefPubMed

13.

Maetzel D, Denzel S, Mack B, Canis M, Went P, Benk M, et al. Nuclear signalling by tumour-associated antigen EpCAM. Nat Cell Biol. 2009;11(2):162–71. https://doi.org/10.1038/ncb1824.CrossRefPubMed

14.

Fagotto F, Aslemarz A. EpCAM cellular functions in adhesion and migration, and potential impact on invasion: a critical review. Biochim Biophys Acta Rev Cancer. 1874;2020(2):188436.CrossRef

15.

Pavsic M, Guncar G, Djinovic-Carugo K, Lenarcic B. Crystal structure and its bearing towards an understanding of key biological functions of EpCAM. Nat Commun. 2014;5(1):4764. https://doi.org/10.1038/ncomms5764.CrossRefPubMed

16.

Molina F, Bouanani M, Pau B, Granier C. Characterization of the type-1 repeat from thyroglobulin, a cysteine-rich module found in proteins from different families. Eur J Biochem. 1996;240(1):125–33. https://doi.org/10.1111/j.1432-1033.1996.0125h.x.CrossRefPubMed

17.

Lenarcic B, Bevec T. Thyropins--new structurally related proteinase inhibitors. Biol Chem. 1998;379(2):105–11.PubMed

18.

Meh P, Pavsic M, Turk V, Baici A, Lenarcic B. Dual concentration-dependent activity of thyroglobulin type-1 domain of testican: specific inhibitor and substrate of cathepsin L. Biol Chem. 2005;386(1):75–83. https://doi.org/10.1515/BC.2005.010.CrossRefPubMed

19.

Lenarcic B, Krishnan G, Borukhovich R, Ruck B, Turk V, Moczydlowski E. Saxiphilin, a saxitoxin-binding protein with two thyroglobulin type 1 domains, is an inhibitor of papain-like cysteine proteinases. J Biol Chem. 2000;275(20):15572–7. https://doi.org/10.1074/jbc.M001406200.CrossRefPubMed

20.

Sudhan DR, Siemann DW. Cathepsin L targeting in cancer treatment. Pharmacol Ther. 2015;155:105–16. https://doi.org/10.1016/j.pharmthera.2015.08.007.CrossRefPubMedPubMedCentral

21.

Denhardt DT, Greenberg AH, Egan SE, Hamilton RT, Wright JA. Cysteine proteinase cathepsin L expression correlates closely with the metastatic potential of H-ras-transformed murine fibroblasts. Oncogene. 1987;2(1):55–9.PubMed

22.

Gottesman MM, Sobel ME. Tumor promoters and Kirsten sarcoma virus increase synthesis of a secreted glycoprotein by regulating levels of translatable mRNA. Cell. 1980;19(2):449–55. https://doi.org/10.1016/0092-8674(80)90519-X.CrossRefPubMed

23.

Baricos WH, Zhou Y, Mason RW, Barrett AJ. Human kidney cathepsins B and L. characterization and potential role in degradation of glomerular basement membrane. Biochem J. 1988;252(1):301–4. https://doi.org/10.1042/bj2520301.CrossRefPubMedPubMedCentral

24.

Chambers AF, Colella R, Denhardt DT, Wilson SM. Increased expression of cathepsins L and B and decreased activity of their inhibitors in metastatic, ras-transformed NIH 3T3 cells. Mol Carcinog. 1992;5(3):238–45. https://doi.org/10.1002/mc.2940050311.CrossRefPubMed

25.

Colella R, Jackson T, Goodwyn E. Matrigel invasion by the prostate cancer cell lines, PC3 and DU145, and cathepsin L+B activity. Biotech Histochem. 2004;79(3–4):121–7. https://doi.org/10.1080/10520290400010572.CrossRefPubMed

26.

Seeber A, Braicu I, Untergasser G, Nassir M, Fong D, Botta L, et al. Detection of soluble EpCAM (sEpCAM) in malignant ascites predicts poor overall survival in patients treated with catumaxomab. Oncotarget. 2015;6(28):25017–23. https://doi.org/10.18632/oncotarget.4496.CrossRefPubMedPubMedCentral

27.

Seeber A, Martowicz A, Spizzo G, Buratti T, Obrist P, Fong D, et al. Soluble EpCAM levels in ascites correlate with positive cytology and neutralize catumaxomab activity in vitro. BMC Cancer. 2015;15(1):372. https://doi.org/10.1186/s12885-015-1371-1.CrossRefPubMedPubMedCentral

28.

Zhang W, Yang HC, Wang Q, Yang ZJ, Chen H, Wang SM, et al. Clinical value of combined detection of serum matrix metalloproteinase-9, heparanase, and cathepsin for determining ovarian cancer invasion and metastasis. Anticancer Res. 2011;31(10):3423–8.PubMed

29.

Chen Q, Fei J, Wu L, Jiang Z, Wu Y, Zheng Y, et al. Detection of cathepsin B, cathepsin L, cystatin C, urokinase plasminogen activator and urokinase plasminogen activator receptor in the sera of lung cancer patients. Oncol Lett. 2011;2(4):693–9. https://doi.org/10.3892/ol.2011.302.CrossRefPubMedPubMedCentral

30.

Schnell U, Kuipers J, Mueller JL, Veenstra-Algra A, Sivagnanam M, Giepmans BN. Absence of cell-surface EpCAM in congenital tufting enteropathy. Hum Mol Genet. 2013;22(13):2566–71. https://doi.org/10.1093/hmg/ddt105.CrossRefPubMedPubMedCentral

31.

Pathak SJ, Mueller JL, Okamoto K, Das B, Hertecant J, Greenhalgh L, et al. EPCAM mutation update. Variants associated with congenital tufting enteropathy and lynch syndrome. Hum Mutat. 2019;40(2):142–61. https://doi.org/10.1002/humu.23688.CrossRefPubMed

32.

Tate JG, Bamford S, Jubb HC, Sondka Z, Beare DM, Bindal N, et al. COSMIC: the catalogue of somatic mutations in Cancer. Nucleic Acids Res. 2019;47(D1):D941–7. https://doi.org/10.1093/nar/gky1015.CrossRef

33.

Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012;2(5):401–4. https://doi.org/10.1158/2159-8290.CD-12-0095.CrossRef

34.

Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7(4):248–9. https://doi.org/10.1038/nmeth0410-248.CrossRefPubMedPubMedCentral

35.

Werle B, Staib A, Julke B, Ebert W, Zladoidsky P, Sekirnik A, et al. Fluorometric microassays for the determination of cathepsin L and cathepsin S activities in tissue extracts. Biol Chem. 1999;380(9):1109–16. https://doi.org/10.1515/BC.1999.138.CrossRefPubMed

36.

Yang Z, Cox JL. Cathepsin L increases invasion and migration of B16 melanoma. Cancer Cell Int. 2007;7(1):8. https://doi.org/10.1186/1475-2867-7-8.CrossRefPubMedPubMedCentral

37.

Felbor U, Dreier L, Bryant RA, Ploegh HL, Olsen BR, Mothes W. Secreted cathepsin L generates endostatin from collagen XVIII. EMBO J. 2000;19(6):1187–94. https://doi.org/10.1093/emboj/19.6.1187.CrossRefPubMedPubMedCentral

38.

Dykes SS, Fasanya HO, Siemann DW. Cathepsin L secretion by host and neoplastic cells potentiates invasion. Oncotarget. 2019;10(53):5560–8. https://doi.org/10.18632/oncotarget.27182.CrossRefPubMedPubMedCentral

39.

Schnell U, Kuipers J, Giepmans BN. EpCAM proteolysis: new fragments with distinct functions? Biosci Rep. 2013;33(2):e00030. https://doi.org/10.1042/BSR20120128.CrossRefPubMedPubMedCentral

40.

Han ML, Zhao YF, Tan CH, Xiong YJ, Wang WJ, Wu F, et al. Cathepsin L upregulation-induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells. Acta Pharmacol Sin. 2016;37(12):1606–22. https://doi.org/10.1038/aps.2016.93.CrossRefPubMedPubMedCentral

41.

Novinec M, Kordis D, Turk V, Lenarcic B. Diversity and evolution of the thyroglobulin type-1 domain superfamily. Mol Biol Evol. 2006;23(4):744–55. https://doi.org/10.1093/molbev/msj082.CrossRefPubMed

42.

Mihelic M, Turk D. Two decades of thyroglobulin type-1 domain research. Biol Chem. 2007;388(11):1123–30. https://doi.org/10.1515/BC.2007.155.CrossRefPubMed

43.

Palermo C, Joyce JA. Cysteine cathepsin proteases as pharmacological targets in cancer. Trends Pharmacol Sci. 2008;29(1):22–8. https://doi.org/10.1016/j.tips.2007.10.011.CrossRefPubMed

44.

Lankelma JM, Voorend DM, Barwari T, Koetsveld J, Van der Spek AH, De Porto AP, et al. Cathepsin L, target in cancer treatment? Life Sci. 2010;86(7–8):225–33. https://doi.org/10.1016/j.lfs.2009.11.016.CrossRefPubMed

45.

Sui H, Shi C, Yan Z, Wu M. Overexpression of Cathepsin L is associated with chemoresistance and invasion of epithelial ovarian cancer. Oncotarget. 2016;7(29):45995–6001. https://doi.org/10.18632/oncotarget.10276.CrossRefPubMedPubMedCentral

46.

Joyce JA, Baruch A, Chehade K, Meyer-Morse N, Giraudo E, Tsai FY, et al. Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell. 2004;5(5):443–53. https://doi.org/10.1016/S1535-6108(04)00111-4.CrossRefPubMed

47.

Bell-McGuinn KM, Garfall AL, Bogyo M, Hanahan D, Joyce JA. Inhibition of cysteine cathepsin protease activity enhances chemotherapy regimens by decreasing tumor growth and invasiveness in a mouse model of multistage cancer. Cancer Res. 2007;67(15):7378–85. https://doi.org/10.1158/0008-5472.CAN-07-0602.CrossRefPubMed

48.

van der Gun BT, de Groote ML, Kazemier HG, Arendzen AJ, Terpstra P, Ruiters MH, et al. Transcription factors and molecular epigenetic marks underlying EpCAM overexpression in ovarian cancer. Br J Cancer. 2011;105(2):312–9. https://doi.org/10.1038/bjc.2011.231.CrossRefPubMedPubMedCentral

49.

Soysal SD, Muenst S, Barbie T, Fleming T, Gao F, Spizzo G, et al. EpCAM expression varies significantly and is differentially associated with prognosis in the luminal B HER2(+), basal-like, and HER2 intrinsic subtypes of breast cancer. Br J Cancer. 2013;108(7):1480–7. https://doi.org/10.1038/bjc.2013.80.CrossRefPubMedPubMedCentral

50.

Ralhan R, Cao J, Lim T, Macmillan C, Freeman JL, Walfish PG. EpCAM nuclear localization identifies aggressive thyroid cancer and is a marker for poor prognosis. BMC Cancer. 2010;10(1):331. https://doi.org/10.1186/1471-2407-10-331.CrossRefPubMedPubMedCentral

51.

Paliwal P, Gupta J, Tandon R, Sharma N, Titiyal JS, Kashyap S, et al. Identification and characterization of a novel TACSTD2 mutation in gelatinous drop-like corneal dystrophy. Mol Vis. 2010;16:729–39.PubMedPubMedCentral

52.

Trebak M, Begg GE, Chong JM, Kanazireva EV, Herlyn D, Speicher DW. Oligomeric state of the colon carcinoma-associated glycoprotein GA733-2 (Ep-CAM/EGP40) and its role in GA733-mediated homotypic cell-cell adhesion. J Biol Chem. 2001;276(3):2299–309. https://doi.org/10.1074/jbc.M004770200.CrossRefPubMed

53.

Pavsic M, Lenarcic B. Expression, crystallization and preliminary X-ray characterization of the human epithelial cell-adhesion molecule ectodomain. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011;67(Pt 11):1363–6. https://doi.org/10.1107/S1744309111031897.CrossRefPubMedPubMedCentral

54.

Balzar M, Briaire-de Bruijn IH, Rees-Bakker HA, Prins FA, Helfrich W, de Leij L, et al. Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions. Mol Cell Biol. 2001;21(7):2570–80. https://doi.org/10.1128/MCB.21.7.2570-2580.2001.CrossRefPubMedPubMedCentral

55.

Blum G, von Degenfeld G, Merchant MJ, Blau HM, Bogyo M. Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes. Nat Chem Biol. 2007;3(10):668–77. https://doi.org/10.1038/nchembio.2007.26.CrossRefPubMed

56.

Sadaghiani AM, Verhelst SH, Gocheva V, Hill K, Majerova E, Stinson S, et al. Design, synthesis, and evaluation of in vivo potency and selectivity of epoxysuccinyl-based inhibitors of papain-family cysteine proteases. Chem Biol. 2007;14(5):499–511. https://doi.org/10.1016/j.chembiol.2007.03.010.CrossRefPubMed

Title: Cancer-associated mutations reveal a novel role for EpCAM as an inhibitor of cathepsin-L and tumor cell invasion
Authors: Narendra V. Sankpal
Taylor C. Brown
Timothy P. Fleming
John M. Herndon
Anusha A. Amaravati
Allison N. Loynd
William E. Gillanders
Publication date: 01-12-2021
Publisher: BioMed Central
Published in: BMC Cancer / Issue 1/2021
Electronic ISSN: 1471-2407
DOI: https://doi.org/10.1186/s12885-021-08239-z

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