Signal Transducer and Activator of Transcription (Stat)-3 Activates the Receptor Tyrosine Kinase-Like Orphan (ROR)-1 Gene in Chronic Lymphocytic Leukemia (CLL).

Abstract 57

ROR1, an embryonic protein involved in organogenesis and Wnt signaling, is expressed in B-cell CLL. Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors ψ-interferon activation sequence (GAS)-like elements typically activated by Stat3, we sought to determine whether Stat3 activates ROR1. In MM1 cells interleukin (IL)-6 induced Stat3 phosphorylation and upregulated ROR1 whereas STAT3-siRNA downregulated both Stat3 and ROR1 protein levels, suggesting that Stat3 transcribes ROR1. Therefore, we cloned the human ROR1 promoter, generated a series of truncated promoter constructs and assessed their activity by using the luciferase assay. We found that IL-6 augmented the luciferase activity of ROR1 -195, ROR1 -666, ROR1 -834, and co-transfection with Stat3-siRNA significantly attenuated it, suggesting that IL-6 enhanced ROR1 expression by activating Stat3. Furthermore, we established that a region, located between bp -122 and -134, harbors a GAS-like element and activates the ROR1 promoter upon exposure to IL-6. Binding of Stat3 to that region in IL-6-stimulated MM1 cells was confirmed by the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). To test whether Stat3 transcribes ROR1 in CLL, we obtained fresh CLL cells and by using the same GAS-like element-containing probe we performed EMSA. CLL cell nuclear protein bound this probe and anti-Stat3 and -phsophoserine Stat3 antibodies induced a super-shift. CLL cell ChIP confirmed that Stat3 binds to the promoter of ROR1 as well as the promoters of the Stat3-regulated genes STAT3, c-Myc and P21, but not that of the control gene RPL30. Finally, using qRT-PCR and western immunoblotting we determined that STAT3-shRNA downregulated ROR1, STAT3 and STAT3-regulated gene mRNA by 4-6 fold, and Stat3 and ROR1 protein levels by 50%. Taken together, these data suggest that constitutively activated Stat3 binds to the ROR1 promoter, activates transcription, and induces production of ROR1 protein in CLL cells.