Journal of Cancer Research and Clinical Oncology

, Volume 141, Issue 9, pp 1563–1574 | Cite as

Role of tumor cell surface lysosome-associated membrane protein-1 (LAMP1) and its associated carbohydrates in lung metastasis

  • Akhil Kumar Agarwal
  • Nithya Srinivasan
  • Rashmi Godbole
  • Shyam K. More
  • Srikanth Budnar
  • Rajiv P. Gude
  • Rajiv D. KalraiyaEmail author
Original Article – Cancer Research



Expression of lysosome-associated membrane protein-1 (LAMP1) on the surface correlates with metastatic potential of B16 melanoma cells. Downregulation of their expression in high metastatic (B16F10) cells reduced their surface expression and metastatic potential. Present investigations explore if overexpression of LAMP1 on the surface of low metastatic (B16F1) cells augment their metastatic ability, and if so, how?


B16F1 cells were transduced with lentiviral vector carrying mutant-LAMP1 (Y386A) (mutLAMP1). Surface expression of LAMP1 and carbohydrates was analyzed by flow cytometry, immunofluorescence and/or immunoprecipitation and Western blotting. Cell spreading and motility were assessed on components of extracellular matrix (ECM) (fibronectin) and basement membrane (BM) (matrigel), and galectin-3-coated coverslips/plates. Metastatic potential was assessed using experimental metastasis assay.


Pre-incubation with anti-LAMP1 antibodies significantly reduced lung metastasis of B16F10 cells. Overexpression of mutLAMP1 significantly increased its surface expression on B16F1 cells, resulting in increased cellular spreading and motility on fibronectin and matrigel. LAMP1 is the major carrier of poly-N-acetyllactosamine (polyLacNAc) on B16F10 cells. However, significantly higher expression of mutLAMP1 had no effect on galectin-3 binding on cell surface or on spreading or motility of cells on galectin-3-coated coverslips/plates. These cells also failed to show any gain in metastatic ability. This could be because LAMP1 from these cells carried significantly lower levels of polyLacNAc in comparison with B16F10 cells.


PolyLacNAc on B16F10 cells and galectin-3 on lungs are the major participants in melanoma metastasis. Although surface LAMP1 promotes interactions with organ ECM and BM, carbohydrates on LAMP1 play a decisive role in dictating lung metastasis.


Cell surface LAMP1 Organ-specific metastasis β1,6 branched N-oligosaccharides Poly-N-acetyllactosamine Galectin-3 Motility 



We thank Dr. Hakon Leffler, Lund University, Sweden, for the expression vector for rhgalectin-3 and National Centre for Cell Science, Pune, for the melanoma cell lines. We acknowledge the help extended by Mrs. Vaishali Kailaje, Mrs. Tanuja Durve, Mrs. Mansi Samarth and Mr. Jayraj Kasale for laser confocal and inverted microscopy, Mrs. Rekha Gour and Ms. Shamal Vetale for flow cytometry, Mr. D. S. Chavan and Mr. A. M. Pawar for technical help and Mr. Sanjay Bane for the help in experimental metastasis and immunoprecipitation experiments. We acknowledge the financial assistance in the form of Senior Research Fellowship to Mr. Akhil Kumar Agarwal, Ms. Nithya Srinivasan and Mr. Shyam K, and more from Council for Scientific and Industrial Research (CSIR), Government of India and Department of Biotechnology (DBT), Government of India for funding the project.

Conflict of interest

We declare that we have no conflict of interest.

Supplementary material

432_2015_1917_MOESM1_ESM.tif (2.9 mb)
Overexpression of wtLAMP1 in B16F1 cells has no effect on surface expression of LAMP1 as well as spreading of melanoma cells on fibronectin. a Comparison of surface expression of LAMP1 by flow cytometry, in B16F1 cells infected with viruses having empty vector (VC) or those having either wtLAMP1 (WT) or mutLAMP1 (C1 and C11) and F10 cells. b Immunofluorescence images of the B16F1 cells infected with viruses having empty vector (VC) or those having either wtLAMP1 (WT) or mutLAMP1 (C1) stained with anti-LAMP1 antibody and FITC labelled secondary antibody (green). c Spreading of the same cells on fibronectin (FN) coated coverslips as assessed by staining with Phalloidin-FITC (green). DAPI was used to stain the nuclei (blue). Scale bar = 5 μm (TIFF 2924 kb)


  1. Agarwal AK, Kalraiya RD (2014) Glycosylation regulates the expression of Lysosome Associated Membrane Protein-1 (LAMP1) on the cell surface. J Biosci Technol 5:556–563Google Scholar
  2. Agarwal AK, Gude RP, Kalraiya RD (2014) Regulation of melanoma metastasis to lungs by cell surface Lysosome Associated Membrane Protein-1 (LAMP1) via galectin-3. Biochem Biophys Res Commun 449:332–337CrossRefPubMedGoogle Scholar
  3. Alter G, Malenfant JM, Altfeld M (2004) CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods 294:15–22CrossRefPubMedGoogle Scholar
  4. Bayer EA, Wilchek M (1990) Protein biotinylation. Methods Enzymol 184:138–160CrossRefPubMedGoogle Scholar
  5. Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, Koup RA (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281:65–78CrossRefPubMedGoogle Scholar
  6. Bretscher A, Edwards K, Fehon RG (2002) ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 3:586–599CrossRefPubMedGoogle Scholar
  7. Brooks SA, Lomax-Browne HJ, Carter TM, Kinch CE, Hall D (2010) Molecular interactions in cancer cell metastasis. Acta Histochem 112:3–25CrossRefPubMedGoogle Scholar
  8. Chakraborty AK et al (2001) Fusion hybrids with macrophage and melanoma cells up-regulate N-acetylglucosaminyltransferase V, β1-6 branching, and metastasis. Cell Growth Differ 12:623–630PubMedGoogle Scholar
  9. Cohnen A et al (2013) Surface CD107a/LAMP-1 protects natural killer cells from degranulation-associated damage. Blood 122:1411–1418CrossRefPubMedGoogle Scholar
  10. Dange MC et al (2014) Galectin-3 expressed on different lung compartments promotes organ specific metastasis by facilitating arrest, extravasation and organ colonization via high affinity ligands on melanoma cells. Clin Exp Metastasis 31:661–673CrossRefPubMedGoogle Scholar
  11. Dennis JW, Laferte S, Waghorne C, Breitman ML, Kerbel RS (1987) Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 236:582–586CrossRefPubMedGoogle Scholar
  12. Febbraio M, Silverstein R (1990) Identification and characterization of LAMP-1 as an activation-dependent platelet surface glycoprotein. J Biol Chem 265:18531–18537PubMedGoogle Scholar
  13. Federici C et al (2009) Pleiotropic function of ezrin in human metastatic melanomas. Int J Cancer 124:2804–2812CrossRefPubMedGoogle Scholar
  14. Fidler IJ (2003) The pathogenesis of cancer metastasis: the ‘seed and soil’hypothesis revisited. Nat Rev Cancer 3:453–458CrossRefPubMedGoogle Scholar
  15. Fukuda M (1991) Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking. J Biol Chem 266:21327–21330PubMedGoogle Scholar
  16. Garrigues J, Anderson J, Hellstrom K, Hellstrom I (1994) Anti-tumor antibody BR96 blocks cell migration and binds to a lysosomal membrane glycoprotein on cell surface microspikes and ruffled membranes. J Cell Biol 125:129–142CrossRefPubMedGoogle Scholar
  17. Gupta GP, Massagué J (2006) Cancer metastasis: building a framework. Cell 127:679–695CrossRefPubMedGoogle Scholar
  18. Hart IR, Fidler IJ (1980) Role of organ selectivity in the determination of metastatic patterns of B16 melanoma. Cancer Res 40:2281–2287PubMedGoogle Scholar
  19. Häuselmann I, Borsig L (2014) Altered tumor-cell glycosylation promotes metastasis. Front Oncol 4:28PubMedCentralCrossRefPubMedGoogle Scholar
  20. Heffernan M, Yousefi S, Dennis JW (1989) Molecular characterization of P2B/LAMP-1, a major protein target of a metastasis-associated oligosaccharide structure. Cancer Res 49:6077–6084PubMedGoogle Scholar
  21. Inohara H, Raz A (1994) Identification of human melanoma cellular and secreted ligands for galectin-3. Biochem Biophys Res Commun 201:1366–1375CrossRefPubMedGoogle Scholar
  22. Irmisch A, Huelsken J (2013) Metastasis: new insights into organ-specific extravasation and metastatic niches. Exp Res 319:1604–1610CrossRefGoogle Scholar
  23. Kannan K, Stewart RM, Bounds W, Carlsson SR, Fukuda M, Betzing KW, Holcombe RF (1996) Lysosome-associated membrane proteins h-LAMP1 (CD107a) and h-LAMP2 (CD107b) are activation-dependent cell surface glycoproteins in human peripheral blood mononuclear cells which mediate cell adhesion to vascular endothelium. Cell Immunol 171:10–19CrossRefPubMedGoogle Scholar
  24. Krishnan V, Bane SM, Kawle PD, Naresh KN, Kalraiya RD (2005) Altered melanoma cell surface glycosylation mediates organ specific adhesion and metastasis via lectin receptors on the lung vascular endothelium. Clin Exp Metastasis 22:11–24CrossRefPubMedGoogle Scholar
  25. Kundra R, Kornfeld S (1999) Asparagine-linked oligosaccharides protect Lamp-1 and Lamp-2 from intracellular proteolysis. J Biol Chem 274:31039–31046CrossRefPubMedGoogle Scholar
  26. Laferté S, Dennis JW (1988) Glycosylation-dependent collagen-binding activities of two membrane glycoproteins in MDAY-D2 tumor cells. Cancer Res 48:4743–4748PubMedGoogle Scholar
  27. Lagana A, Goetz JG, Cheung P, Raz A, Dennis JW, Nabi IR (2006) Galectin binding to Mgat5-modified N-glycans regulates fibronectin matrix remodeling in tumor cells. Mol Cell Biol 26:3181–3193PubMedCentralCrossRefPubMedGoogle Scholar
  28. Liu F-T, Rabinovich GA (2005) Galectins as modulators of tumour progression. Nat Rev Cancer 5:29–41CrossRefPubMedGoogle Scholar
  29. Mane SM, Marzella L, Bainton DF, Holt VK, Cha Y, Hildreth JE, August JT (1989) Purification and characterization of human lysosomal membrane glycoproteins. Arch Biochem Biophys 268:360–378CrossRefPubMedGoogle Scholar
  30. McCormick PJ, Bonventre EJ, Finneran A (1998) LAMP-1/ESG p appears on the cell surface of single celled mouse embryos subsequent to fertilization. In Vitro Cell Dev Biol Anim 34:353–355CrossRefPubMedGoogle Scholar
  31. McGary EC, Lev DC, Bar-Eli M (2002) Cellular adhesion pathways and metastatic potential of human melanoma. Cancer Biol Ther 1:454–459CrossRefGoogle Scholar
  32. Neisch AL, Fehon RG (2011) Ezrin, radixin and moesin: key regulators of membrane–cortex interactions and signaling. Curr Opin Cell Biol 23:377–382PubMedCentralCrossRefPubMedGoogle Scholar
  33. Nguyen DX, Bos PD, Massagué J (2009) Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer 9:274–284CrossRefPubMedGoogle Scholar
  34. Poste G, Nicolson GL (1980) Arrest and metastasis of blood-borne tumor cells are modified by fusion of plasma membrane vesicles from highly metastatic cells. Proc Natl Acad Sci 77:399–403PubMedCentralCrossRefPubMedGoogle Scholar
  35. Ranjan A, Kalraiya RD (2013) α2, 6 Sialylation associated with increased β1, 6-branched N-oligosaccharides influences cellular adhesion and invasion. J Biosci 38:867–876CrossRefPubMedGoogle Scholar
  36. Ranjan A, Bane SM, Kalraiya RD (2014) Glycosylation of the laminin receptor (α3β1) regulates its association with tetraspanin CD151: impact on cell spreading, motility, degradation and invasion of basement membrane by tumor cells. Exp Cell Res 322:249–264CrossRefPubMedGoogle Scholar
  37. Reddy B, Kalraiya RD (2006) Sialilated β1, 6 branched N-oligosaccharides modulate adhesion, chemotaxis and motility of melanoma cells: effect on invasion and spontaneous metastasis properties. Biochim Biophys Acta 1760:1393–1402CrossRefPubMedGoogle Scholar
  38. Saitoh O, Wang W, Lotan R, Fukuda M (1992) Differential glycosylation and cell surface expression of lysosomal membrane glycoproteins in sublines of a human colon cancer exhibiting distinct metastatic potentials. J Biol Chem 267:5700–5711PubMedGoogle Scholar
  39. Sarafian V et al (1998) Expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in human tumor cells. Int J Cancer 75:105–111CrossRefPubMedGoogle Scholar
  40. Sawada R, Lowe J, Fukuda M (1993) E-selectin-dependent adhesion efficiency of colonic carcinoma cells is increased by genetic manipulation of their cell surface lysosomal membrane glycoprotein-1 expression levels. J Biol Chem 268:12675–12681PubMedGoogle Scholar
  41. Sehgal L, Budnar S, Bhatt K, Sansare S, Mukhopadhaya A, Kalraiya RD, Dalal SN (2012) Generation of HIV-1 based bi-cistronic lentiviral vectors for stable gene expression and live cell imaging. Indian J Exp Biol 50:669–676PubMedGoogle Scholar
  42. Srinivasan N, Bane SM, Ahire SD, Ingle AD, Kalraiya RD (2009) Poly N-acetyllactosamine substitutions on N-and not O-oligosaccharides or Thomsen-Friedenreich antigen facilitate lung specific metastasis of melanoma cells via galectin-3. Glycoconj J 26:445–456CrossRefPubMedGoogle Scholar
  43. Tomlinson J, Wang JL, Barsky SH, Lee MC, Bischoff J, Nguyen M (2000) Human colon cancer cells express multiple glycoprotein ligands for E-selectin. Int J Oncol 16:347–353PubMedGoogle Scholar
  44. Valastyan S, Weinberg RA (2011) Tumor metastasis: molecular insights and evolving paradigms. Cell 147:275–292PubMedCentralCrossRefPubMedGoogle Scholar
  45. Weiss L (1990) Metastatic inefficiency. Adv Cancer Res 54:159–211CrossRefPubMedGoogle Scholar
  46. Weiss L (1992) Comments on hematogenous metastatic patterns in humans as revealed by autopsy. Clin Exp Metastasis 10:191–199CrossRefPubMedGoogle Scholar
  47. Williams MA, Fukuda M (1990) Accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail. J Cell Biol 111:955–966CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Akhil Kumar Agarwal
    • 1
  • Nithya Srinivasan
    • 1
  • Rashmi Godbole
    • 2
  • Shyam K. More
    • 1
  • Srikanth Budnar
    • 3
  • Rajiv P. Gude
    • 1
  • Rajiv D. Kalraiya
    • 1
    Email author
  1. 1.Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)Tata Memorial Centre, Sector 22Kharghar, Navi MumbaiIndia
  2. 2.Proteomics Lab, Biochemical Sciences DivisionCSIR-National Chemical LaboratoryPuneIndia
  3. 3.Division of Molecular Cell Biology, Institute for Molecular BioscienceThe University of QueenslandBrisbaneAustralia

Personalised recommendations