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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. Kalraiya
Original Article – Cancer Research

Abstract

Purpose

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?

Methods

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.

Results

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.

Conclusions

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.

Keywords

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

Notes

Acknowledgments

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)

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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
  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

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