Thrombospondin-1 receptor mediates autophagy of RAS-expressing cancer cells and triggers tumour growth inhibition.

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.

Materials And 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.

Conclusion: 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.