FGR Src Family Kinase Causes Signaling and Phenotypic Shift Mimicking Retinoic Acid-Induced Differentiation of Leukemic Cells

Abstract

ABSTRACTRetinoic acid (RA) is an embryonic morphogen used in cancer differentiation-therapy. It causes a plethora of changes in gene expression culminating in cell differentiation. We now find that amongst them, expression of the Src-family-kinase, FGR, by itself causes cell differentiation analogous to RA. The historically dominant/classical paradigm for RA mechanism of action is transcriptional activation via binding to the ligand-activated nuclear receptors, RAR/RXR. In the HL-60 human myelo-monocytic leukemia model, an actively proliferating, phenotypically immature, lineage bi-potent NCI-60 cell line, RA causes election of the myeloid lineage and phenotypic maturation with G1/0 growth inhibition. It thereby converts transformed immature proliferating tumor cells to mature growth retarded cells that bear fidelity to non-transformed mature myeloid cells. The present study finds that expression of the FGR SFK(SRC-family-kinase) alone is sufficient to induce differentiation. Akin to RA, the phenotypic conversion manifests as expression of CD38, CD11b, and ROS, as well as the p27(kip1) CDKI (cyclin-dependent-kinase-inhibitor that retards cells in G1/0) characteristic of mature myeloid cells. To pursue mechanistic insight, signaling attributes known to promote RA-induced differentiation were analyzed to see what FGR affected. RA is known to cause expression of FGR which is incorporated into and activates a putative novel cytosolic macromolecular signaling machine(signalsome) that propels differentiation. RA enhances the abundance of signalsome constituents, their associations, and their phosphorylation. The signalsome contains connected nodes that appear as a spine to which the other components are connected. The apparent “nodes” are RAF, LYN, FGR, SLP-76 and CBL. All of these become enriched in the nucleus after RA-treatment. NUMB and VAV appear to provide further scaffolding functions enhanced by RA. RAF in the nucleus complexes with a RARE (retinoic acid-response-element) in the promoter of the blr1 gene, which encodes a serpentine G-protein-coupled-receptor. blr1 transcriptional activation by RA depends on RAF binding. BLR1 expression is necessary to propel RA-induced differentiation, although by itself is not sufficient to cause phenotypic differentiation. Analyzing this signaling process revealed that expression of FGR mimics RA-induced enhancement of the signalsome nodes, enhancing expression of RAF and its phosphorylation, and causing BLR1 expression. Interestingly, for cd38 and blr1, FGR apparently causes expression of genes targeted by RAR/RXR even without RA. FGR thus appears to cause signaling events and phenotypic shift characteristic of RA. In sum, the data indicate that FGR is the “trigger” for RA-induced differentiation. Given the historical perception of FGR as a pro-proliferation, transforming-viral-oncogene, this is a surprising paradigm shift.