ITD‐ and FL‐induced FLT3 signal transduction leads to increased C/EBPβ‐LIP expression and LIP/LAP ratio by different signalling modules

Abstract

Summary FLT3 receptor‐associated signalling plays a role in proliferation and leukaemia. The transcription factor C/EBPβ may be involved in malignancy with its alternative translation product C/EBPβ‐LIP. We investigated a potential connection between FLT3 signalling and the C/EBPβ system in FLT3‐internal tandem duplication (ITD)‐positive leukaemia cells and FLT3‐ITD‐ or FLT3‐wild type (WT)‐transfected 32D cells. In FLT3‐ITD‐positive cells or when ITD sequences were inserted into the FLT3‐WT receptor, significant LIP levels, increased LIP/LAP ratios, and enhanced proliferation rates were detected, which were reduced by FLT3 inhibition. In FLT3‐WT cells, incubation with FLT3 receptor ligand (FL) also elevated LIP, LIP/LAP, and proliferation, albeit to a lesser extent. CEBPB‐directed siRNA decreased both LIP and proliferation rates in FLT3‐ITD‐positive and FL‐stimulated FLT3‐WT‐positive cells. PI3K inhibition affected ITD‐associated and FL‐induced LIP levels. Rapamycin, an inhibitor of mTOR involved in CEBPB translation, completely blocked the increase in LIP in FL‐stimulated FLT3‐WT‐ but not FLT3‐ITD‐positive cells. In contrast, the ITD‐associated LIP elevation was mediated by p90‐ribosomal‐S6‐kinase. This is the first report showing a LIP increase in the presence of ITD or following FL exposure. Our data suggest fundamental differences in the signalling cascades activated via ITD mutations or following FL stimulation, indicating the need for adapted molecular therapy.

CCAAT/Enhancer-binding Protein β Inhibits Proliferation in Monocytic Cells by Affecting the Retinoblastoma Protein/E2F/Cyclin E Pathway but Is Not Directly Required for Macrophage Morphology

“…Cell lysis, total RNA isolation, validation of RNA concentration/purity, and generation of external standard curves are described elsewhere (24). Two μg of total RNA were reverse-transcribed as described previously (25).…”

“…Expression of human IL-8 mRNA was assessed by real-time RT-PCR using a LightCycler 480, universal probe library probe 72 (Roche Applied Science), and specific primers (5′-AGACAGCAGAGCACACAAGC-3′ and 5′-ATGGTTCCTTCCGGTGGT-3′). For the amplification, a standard protocol for experiments using universal probe library probes was used (24). Normalization of cDNA amounts was performed as described (19).…”

“…Subsequently, the LAP/LIP ratio was calculated by dividing the combined LAP* and LAP value (denoted as LAP in the LAP/LIP ratio) by the respective LIP value (24). Equivalently, the p42/p30 ratio was calculated by dividing the p42 value by the respective p30 value.…”

The shortest isoform of C/EBPβ, liver inhibitory protein (LIP), collaborates with Evi1 to induce AML in a mouse BMT model

“…28 Furthermore, expression of LIP is increased in FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD)-positive myeloid leukemic cells. 29 On the other hand, C/EBPb was shown to be upregulated during ATRAinduced differentiation of acute promyelocytic leukemia cells, and it was considered as a potential therapeutic target in leukemia for granulocytic differentiation. 30 Taken together, C/EBPb has leukemia suppressor-like activity in acute promyelocytic leukemia, and it also has critical oncogenic functions in FLT3-ITD leukemia, B-lineage acute lymphoblastic leukemia, and multiple myeloma.…”

Epidermal growth factor-induced C/EBPbeta participates in EMT by dampening miR-203 in esophageal squamous cell carcinoma

“…Subsequently, the LIP:LAP ratio was calculated by dividing the LIP value by the respective combined LAP1 and LAP2 value (together denoted as LAP in the LIP:LAP ratio) as previously described (Haas et al, 2010).…”