title: Thrombospondin-1 receptor mediates autophagy of RAS-expressing cancer cells and triggers tumour growth inhibition.
abstract: BACKGROUND
  The anti-angiogenic activity of thrombospondin-1 (TSP-1) is suppressed in cancer cells, fact which has generated considerable interest in generating the respective therapeutic mimetics. These efforts were almost exclusively centered on peptides targeting CD36, the known TSP-1 receptor. Since the effects of these agents were less dramatic than those of full-length. TSP-1 questions could be raised about the cancer-related roles of additional TSP-1 domains and receptors, such as CD47, which bind the C-terminal sequences of this protein.
  
  
  METHODS
  The MDFB6 and B6ras cell lines were treated with the anti-CD47 antibody or C-terminal TSP-1 peptide 4N1K and cell viability was monitored using Aqueous Non-Radioactive Cell Proliferation Assay, DNA fragmentation, caspase-3 activation, and cell membrane depolarization assays. The cells were also stained with acridine orange and the anti-LC3 antibody to detect autophagy. B6ras tumours were generated in SCID-NOD mice and tumour responses to injections of the 4N1K peptide were recorded over time.
  
  
  RESULTS
  We demonstrated a selective loss of viability of RAS-transformed cells upon ligation of the CD47 receptor. Affected cells did not exhibit hallmarks of apoptosis, but instead were stained with acridine orange and exhibited a punctuate pattern of immunoreactivity for LC3, both features of autophagy. The 4N1K peptide administration also caused a modest but specific and significant tumour growth inhibition in vivo.
  
  
  CONCLUSIONS
  Our study offers an additional mechanism whereby TSP-1 affects tumour cells directly, in an angiogenesis-independent and CD47-mediated manner. This is consistent with near complete loss of TSP-1 expression in RAS-transformed cells and may open new avenues for therapies involving TSP-1 peptides, in a subset of tumours.
authors.affiliation: Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. kalas@iitd.pan.wroc.pl
authors.forename: Wojciech
authors.initials: W
authors.lastname: Kalas
authors.forename: Ewelina
authors.initials: E
authors.lastname: Swiderek
authors.forename: Marta
authors.initials: M
authors.lastname: Switalska
authors.forename: Joanna
authors.initials: J
authors.lastname: Wietrzyk
authors.forename: Janusz
authors.initials: J
authors.lastname: Rak
authors.forename: Leon
authors.initials: L
authors.lastname: Strzadala
mesh_terms: D000818:Animals; D001343:Autophagy; D015153:Blotting, Western; D018955:CD36 Antigens / Q000378:metabolism*; D051928:CD47 Antigen / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry; D011905:Genes, ras / Q000502:physiology*; D007124:Immunoenzyme Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000494:pharmacology; D012878:Skin Neoplasms / Q000235:genetics / Q000473:pathology*; D014407:Tumor Cells, Cultured
chemical_list: D018955:CD36 Antigens; D051928:CD47 Antigen; D010446:Peptide Fragments; D053148:Caspase 3