Skip to main content

Advertisement

SpringerLink
Log in

SNX3-dependent regulation of epidermal growth factor receptor (EGFR) trafficking and degradation by aspirin in epidermoid carcinoma (A-431) cells

  • Research Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

Since being introduced globally as aspirin in 1899, acetylsalicylic acid has been widely used as an analgesic, anti-inflammation, anti-pyretic, and anti-thrombotic drug for years. Aspirin had been reported to down-regulate surface expression of CD40, CD80, CD86, and MHCII in myeloid dendritic cells (DC), which played essential roles in regulating the immune system. We hypothesized that the down-regulation of these surface membrane proteins is partly due to the ability of aspirin in regulating trafficking/sorting of endocytosed surface membrane proteins. By using an established epidermoid carcinoma cell line (A-431), which overexpresses the epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR), we show that aspirin (1) reduces cell surface expression of EGFR and (2) accumulates endocytosed-EGFR and -TfnR in the early/sorting endosome (ESE). Further elucidation of the mechanism suggests that aspirin enhances recruitment of SNX3 and SNX5 to membranes and consistently, both SNX3 and SNX5 play essential roles in the aspirin-mediated accumulation of endocytosed-TfnR at the ESE. This study sheds light on how aspirin may down-regulate surface expression of EGFR by inhibiting/delaying the exit of endocytosed-EGFR from the ESE and recycling of endocytosed-EGFR back to the cell surface.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

EGFR:

Epidermal growth factor receptor

TfnR:

Transferrin receptor

EE:

Early endosome

ESE:

Early/Sorting endosomes

RE:

Recycling endosome

LE:

Late endosome

SNX:

Sorting nexin

AMC:

Aspirin-induced membrane compartment

NRAMP2:

Natural resistance-associated macrophage protein 2

CysLT1 :

Cysteinyl leukotriene receptor

SR-BI:

Scavenger receptor class B type I

References

  1. Wieduwilt MJ, Moasser MM (2008) The epidermal growth factor receptor family: biology driving targeted therapeutics. Cell Mol Life Sci 65:1566–1584

    Article  PubMed  CAS  Google Scholar 

  2. Sorkin A, Goh LK (2009) Endocytosis and intracellular trafficking of ErbBs. Exp Cell Res 315:683–696

    Article  PubMed  CAS  Google Scholar 

  3. Sadowski L, Pilecka I, Miaczynska M (2009) Signaling from endosomes: location makes a difference. Exp Cell Res 315:1601–1609

    Article  PubMed  CAS  Google Scholar 

  4. Lawrence CM, Ray S, Babyonyshev M, Galluser R, Borhani DW, Harrison SC (1999) Crystal structure of the ectodomain of human transferrin receptor. Science 286:779–782

    Article  PubMed  CAS  Google Scholar 

  5. Gruenheid S, Canonne-Hergaux F, Gauthier S, Hackam DJ, Grinstein S, Gros P (1999) The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes. J Exp Med 189:831–841

    Article  PubMed  CAS  Google Scholar 

  6. Matasic R, Dietz AB, Vuk-Pavlovic S (2000) Cyclooxygenase-independent inhibition of dendritic cell maturation by aspirin. Immunology 101:53–60

    Article  PubMed  CAS  Google Scholar 

  7. Sousa AR, Parikh A, Scadding G, Corrigan CJ, Lee TH (2002) Leukotriene-receptor expression on nasal mucosal inflammatory cells in aspirin-sensitive rhinosinusitis. N Engl J Med 347:1493–1499

    Article  PubMed  CAS  Google Scholar 

  8. Tancevski I, Wehinger A, Schgoer W, Eller P, Cuzzocrea S, Foeger B, Patsch JR, Ritsch A (2006) Aspirin regulates expression and function of scavenger receptor-BI in macrophages: studies in primary human macrophages and in mice. FASEB J 20:1328–1335

    Article  PubMed  CAS  Google Scholar 

  9. Karagiannis TC, Lobachevsky PN, Leung BK, White JM, Martin RF (2006) Receptor-mediated DNA-targeted photoimmunotherapy. Cancer Res 66:10548–10552

    Article  PubMed  CAS  Google Scholar 

  10. Huang D, Cai DT, Chua RY, Kemeny DM, Wong SH (2008) Nitric-oxide synthase 2 interacts with CD74 and inhibits its cleavage by caspase during dendritic cell development. J Biol Chem 283:1713–1722

    Article  PubMed  CAS  Google Scholar 

  11. Ho YH, Cai DT, Huang D, Wang CC, Wong SH (2009) Caspases regulate VAMP-8 expression and phagocytosis in dendritic cells. Biochem Biophys Res Commun 387:371–375

    Article  PubMed  CAS  Google Scholar 

  12. Goding JW, Burns GF (1981) Monoclonal antibody OKT-9 recognizes the receptor for transferrin on human acute lymphocytic leukemia cells. J Immunol 127:1256–1258

    PubMed  CAS  Google Scholar 

  13. Pannecouque C, Daelemans D, De Clercq E (2008) Tetrazolium-based colorimetric assay for the detection of HIV replication inhibitors: revisited 20 years later. Nat Protoc 3:427–434

    Article  PubMed  CAS  Google Scholar 

  14. Ponka P, Lok CN (1999) The transferrin receptor: role in health and disease. Int J Biochem Cell Biol 31:1111–1137

    Article  PubMed  CAS  Google Scholar 

  15. Maxfield FR, McGraw TE (2004) Endocytic recycling. Nat Rev Mol Cell Biol 5:121–132

    Article  PubMed  CAS  Google Scholar 

  16. Le Roy C, Wrana JL (2005) Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat Rev Mol Cell Biol 6:112–126

    Article  PubMed  CAS  Google Scholar 

  17. Fields IC, Shteyn E, Pypaert M, Proux-Gillardeaux V, Kang RS, Galli T, Folsch H (2007) v-SNARE cellubrevin is required for basolateral sorting of AP-1B-dependent cargo in polarized epithelial cells. J Cell Biol 177:477–488

    Article  PubMed  CAS  Google Scholar 

  18. Patki V, Virbasius J, Lane WS, Toh BH, Shpetner HS, Corvera S (1997) Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 94:7326–7330

    Article  PubMed  CAS  Google Scholar 

  19. Wong SH, Xu Y, Zhang T, Hong W (1998) Syntaxin 7, a novel syntaxin member associated with the early endosomal compartment. J Biol Chem 273:275–380

    Google Scholar 

  20. Worby CA, Dixon JE (2002) Sorting out the cellular functions of sorting nexins. Nat Rev Mol Cell Biol 3:919–931

    Article  PubMed  CAS  Google Scholar 

  21. Kurten RC, Cadena DL, Gill GN (1996) Enhanced degradation of EGF receptors by a sorting nexin, SNX1. Science 272:1008–1010

    Article  PubMed  CAS  Google Scholar 

  22. Xu Y, Hortsman H, Seet L, Wong SH, Hong W (2001) SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P. Nat Cell Biol 3:658–666

    Article  PubMed  CAS  Google Scholar 

  23. Takatsu H, Sakurai M, Shin HW, Murakami K, Nakayama K (1998) Identification and characterization of novel clathrin adaptor-related proteins. J Biol Chem 273:24693–24700

    Article  PubMed  CAS  Google Scholar 

  24. Feng Y, Hu J, Xie D, Qin J, Zhong Y, Li X, Xiao W, Wu J, Tao D, Zhang M, Zhu Y, Song Y, Reed E, Li QQ, Gong J (2005) Subcellular localization of caspase-3 activation correlates with changes in apoptotic morphology in MOLT-4 leukemia cells exposed to X-ray irradiation. Int J Oncol 27:699–704

    PubMed  CAS  Google Scholar 

  25. Kalgutkar AS, Crews BC, Rowlinson SW, Garner C, Seibert K, Marnett LJ (1998) Aspirin-like molecules that covalently inactivate cyclooxygenase-2. Science 280:1268–1270

    Article  PubMed  CAS  Google Scholar 

  26. Liyasova MS, Schopfer LM, Lockridge O (2010) Reaction of human albumin with aspirin in vitro: mass spectrometric identification of acetylated lysines 199, 402, 519, and 545. Biochem Pharmacol 79:784–791

    Article  PubMed  CAS  Google Scholar 

  27. Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, Olsen JV, Mann M (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834–840

    Google Scholar 

  28. Chiang N, Serhan CN, Dahlen SE, Drazen JM, Hay DW, Rovati GE, Shimizu T, Yokomizo T, Brink C (2006) The lipoxin receptor ALX: potent ligand-specific and stereoselective actions in vivo. Pharmacol Rev 58:463–487

    Article  PubMed  CAS  Google Scholar 

  29. Claria J, Lee MH, Serhan CN (1996) Aspirin-triggered lipoxins (15-epi-LX) are generated by the human lung adenocarcinoma cell line (A549)-neutrophil interactions and are potent inhibitors of cell proliferation. Mol Med 2:583–596

    PubMed  CAS  Google Scholar 

  30. Piper RC, Katzmann DJ (2007) Biogenesis and function of multivesicular bodies. Ann Rev Cell Dev Biol 23:519–547

    Article  CAS  Google Scholar 

  31. Sorkin A, Von Zastrow M (2002) Signal transduction and endocytosis: close encounters of many kinds. Nat Rev Mol Cell Biol 3:600–614

    Article  PubMed  CAS  Google Scholar 

  32. Kim EK, Choi EJ (2010) Pathological roles of MAPK signaling pathways in human diseases. Biochim Biophys Acta 1802:396–405

    PubMed  CAS  Google Scholar 

  33. Ahn S, Kim J, Lucaveche CL, Reedy MC, Luttrell LM, Lefkowitz RJ, Daaka Y (2002) Src-dependent tyrosine phosphorylation regulates dynamin self-assembly and ligand-induced endocytosis of the epidermal growth factor receptor. J Biol Chem 277:26642–26651

    Article  PubMed  CAS  Google Scholar 

  34. Confalonieri S, Salcini AE, Puri C, Tacchetti C, Di Fiore PP (2000) Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis. J Cell Biol 150:905–912

    Article  PubMed  CAS  Google Scholar 

  35. Mace G, Miaczynska M, Zerial M, Nebreda AR (2005) Phosphorylation of EEA1 by p38 MAP kinase regulates mu opioid receptor endocytosis. EMBO J 24:3235–3246

    Article  PubMed  CAS  Google Scholar 

  36. Oshima T, Miwa H, Joh T (2008) Aspirin induces gastric epithelial barrier dysfunction by activating p38 MAPK via claudin-7. Am J Physiol Cell Physiol 295:C800–C806

    Article  PubMed  CAS  Google Scholar 

  37. Ou YC, Yang CR, Cheng CL, Raung SL, Hung YY, Chen CJ (2007) Indomethacin induces apoptosis in 786-O renal cell carcinoma cells by activating mitogen-activated protein kinases and AKT. Eur J Pharmacol 563:49–60

    Article  PubMed  CAS  Google Scholar 

  38. Becker JC, Muller-Tidow C, Stolte M, Fujimori T, Tidow N, Ilea AM, Brandts C, Tickenbrock L, Serve H, Berdel WE, Domschke W, Pohle T (2006) Acetylsalicylic acid enhances antiproliferative effects of the EGFR inhibitor gefitinib in the absence of activating mutations in gastric cancer. Int J Oncol 29:615–623

    PubMed  CAS  Google Scholar 

  39. Ordan O, Rotem R, Jaspers I, Flescher E (2003) Stress-responsive JNK mitogen-activated protein kinase mediates aspirin-induced suppression of B16 melanoma cellular proliferation. Br J Pharmacol 138:1156–1162

    Article  PubMed  CAS  Google Scholar 

  40. Jana NR (2008) NSAIDs and apoptosis. Cell Mol Life Sci 65:1295–1301

    Article  PubMed  CAS  Google Scholar 

  41. Elwood PC, Gallagher AM, Duthie GG, Mur LA, Morgan G (2009) Aspirin, salicylates, and cancer. Lancet 373:1301–1309

    Article  PubMed  CAS  Google Scholar 

  42. Rothwell PM, Fowkes FG, Belch JF, Ogawa H, Warlow CP, Meade TW (2011) Effect of daily aspirin on long term risk of death due to cancer: analysis of individual patient data from randomised trials. Lancet 377:31–41

    Article  PubMed  CAS  Google Scholar 

  43. Whitnall M, Howard J, Ponka P, Richardson DR (2006) A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics. Proc Natl Acad Sci USA 103:14901–14906

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Dr. Boon Chuan Low for reading the manuscript and his valuable comments. This work was supported by research grants from the Academic Research Council-Ministry of Education, Singapore (R182-000-099-112 to S.H.W) and the Singapore National Medical Research Council (R182-000-137-213 to S.H.W).

Author information

Authors and Affiliations

  1. Immunology Programme, Laboratory of Membrane Trafficking and Immunoregulation, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597, Republic of Singapore

    Kher Hsin Chiow, Yingrou Tan, Rong Yuan Chua, Dachuan Huang & Siew Heng Wong

  2. Laboratory of Flavivirus, National University of Singapore, Singapore, Republic of Singapore

    Mah Lee Mary Ng

  3. Department of Microbiology, National University of Singapore, Singapore, Republic of Singapore

    Kher Hsin Chiow, Yingrou Tan, Rong Yuan Chua, Dachuan Huang, Mah Lee Mary Ng & Siew Heng Wong

  4. Department of Biochemistry, National University of Singapore, Singapore, Republic of Singapore

    Markus R. Wenk

  5. Department of Biological Sciences, National University of Singapore, Singapore, Republic of Singapore

    Markus R. Wenk

  6. Mechanobiology Institute, National University of Singapore, Singapore, Republic of Singapore

    Federico Torta

Authors
  1. Kher Hsin Chiow
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yingrou Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rong Yuan Chua
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dachuan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mah Lee Mary Ng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Federico Torta
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Markus R. Wenk
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Siew Heng Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Siew Heng Wong.

Electronic supplementary material

Below is the link to the electronic supplementary material.

18_2011_887_MOESM1_ESM.tif

Supplementary material 1 (TIFF 895 kb) Supplementary Fig. 1 The aspirin-mediated accumulation of endocytosed cell surface receptors in the ESE is also regulated by the MAPK pathway. (A) Cell extracts from untreated and aspirin-treated A-431 cells were analyzed by Western-blot analysis using antibodies specific for the phosphorylated forms of p38 MAPK, JNK, and ERK. Results are representative of three independent experiments. (B) Surface antibody-TfnR complexes were allowed to be internalized into cells in the absence or presence of aspirin and p38 inhibitor with concentration and duration as indicated. Image, fluorescence microscope; magnification, 100x; white arrow, the compact structure is the ESE/AMC; red arrow, reduced accumulation of eTfnR in the ESE/AMC in the presence of inhibitors and aspirin. Results are representative of at least two independent experiments. (C) Surface antibody-TfnR complexes were allowed to be internalized into A-431 cells in the presence of 10 mM aspirin for 3 h (a) and followed by treatment with 10 mM aspirin (b, j); normal complete media (f, n); aspirin and p38 inhibitor (c, k); aspirin and ERK inhibitor (d, l); aspirin and JNK inhibitor (e, m); p38 inhibitor (g, o); ERK inhibitor (h, p); and JNK inhibitor (l, q) at indicated time points. Image of fluorescence microscope; magnification, 100x; white arrow, the compact structure is the ESE/AMC; red arrow, reduced accumulation of eTfnR in the ESE/AMC in the presence of inhibitors and aspirin; yellow arrow, reduced accumulation of eTfnR in the ESE/AMC upon removal of aspirin and in the presence of inhibitors; purple arrow, reduced accumulation of eTfnR in the ESE/AMC upon removal of aspirin.

18_2011_887_MOESM2_ESM.tif

Supplementary material 2 (TIFF 61 kb) Supplementary Fig. 2 Aspirin inhibits A-431 cell proliferation. A-431 was treated with indicated concentrations of aspirin for 48 h. Cells were subjected to MTT and the number of viable cells in each sample was normalized to untreated sample. Points, average of normalized values from three independent experiments; bars, ±SD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chiow, K.H., Tan, Y., Chua, R.Y. et al. SNX3-dependent regulation of epidermal growth factor receptor (EGFR) trafficking and degradation by aspirin in epidermoid carcinoma (A-431) cells. Cell. Mol. Life Sci. 69, 1505–1521 (2012). https://doi.org/10.1007/s00018-011-0887-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-011-0887-z

Keywords

Search

Navigation