Osteopontin and the C-terminal peptide of thrombospondin-4 compete for CD44 binding and have opposite effects on CD133+ cell colony formation

<p>Abstract</p> <p>Background</p> <p>C21, the C-terminal peptide of thrombospondin-4, has growth promoting activity and was discovered as one of several erythropoietin-dependent endothelial proteins. C21 stimulates red cell formation in anemic mice and is a growth factor for CD34+ and CD36+ hematopoietic cells, skin fibroblasts and kidney epithelial cells. ROD1 has been identified as an intracellular mediator. Nothing is known about the existence of putative C21 receptors on plasma membranes of target cells.</p> <p>Findings</p> <p>We analyzed the nature of C21-binding proteins in cell lysates of skin fibroblasts using C21 affinity columns. The membrane receptor CD44 was identified as C21-binding protein by mass spectrometry. We were unable to demonstrate any direct involvement of CD44 on cell growth or the effect of C21 on cell proliferation. A soluble form of CD44 was synthesized in insect cells and purified from culture supernatants with a combination of PVDF filtration in the presence of ammonium sulphate and HPLC. Both osteopontin and hyaluronic acid competitively displaced Biotin-C21 binding to CD44. In a colony-forming assay using primitive CD133+ hematopoietic stem cells from cord blood, osteopontin and C21 had opposite effects and C21 reduced the inhibitory action of osteopontin.</p> <p>Conclusion</p> <p>CD44 is a C21-binding membrane protein. We could not demonstrate an involvement of CD44 in the proliferative action of C21. Nevertheless, based on the antagonism of C21 and osteopontin in hematopoietic precursors, we speculate that C21 could indirectly have a major impact on hematopoietic stem cell proliferation, by hindering osteopontin membrane binding at the level of the bone marrow niche.</p

The role of eIF4E in response and acquired resistance to vemurafenib in melanoma

In eukaryotic cells, the rate-limiting component for cap-dependent mRNA translation is the translation initiation factor eIF4E. eIF4E is overexpressed in a variety of human malignancies, but whether it has a role in melanoma remains obscure. We hypothesized that eIF4E promotes melanoma cell proliferation and facilitates the development of acquired resistance to the BRAF inhibitor vemurafenib. We show that eIF4E is overexpressed in a panel of melanoma cell lines, compared with immortalized melanocytes. Knockdown of eIF4E significantly repressed the proliferation of a subset of melanoma cell lines. Moreover, in BRAFV600E melanoma cell lines, vemurafenib inhibits 4E-BP1 phosphorylation, thus promoting its binding to eIF4E. Cap-binding and polysome profiling analysis confirmed that vemurafenib stabilizes the eIF4E–4E-BP1 association and blocks mRNA translation, respectively. Conversely, in cells with acquired resistance to vemurafenib, there is an increased dependence on eIF4E for survival; 4E-BP1 is highly phosphorylated and thus eIF4E-4E-BP1 associations are impeded. Moreover, increasing eIF4E activity by silencing 4E-BP1/2 renders vemurafenib-responsive cells more resistant to BRAF inhibition. In conclusion, these data suggest that therapeutically targeting eIF4E may be a viable means of inhibiting melanoma cell proliferation and overcoming vemurafenib resistance