C/EBPβ Expression in ALK+ Anaplastic Large Cell Lymphomas (ALCL) Is Regulated by Stat3 Signaling Pathway.
Authors: Anastasov Natasa , Rudelius Martina , Klier Margit , Dau Therese , Angermeier Daniela , Pittaluga Stefania , Duyster Justus , Fend Falko , Raffeld Mark , Quintanilla-Martinez Leticia 
Published: April 2020

Abstract
Background: ALK+ ALCL is characterized by the t(2;5) chromosomal translocation, resulting in the expression of a fusion protein called NPM-ALK. We recently reported the abnormal expression of the transcription factor C/EBPβ in ALCL, and demonstrated that C/EBPβ expression is dependent on NPM-ALK kinase activity. However, it is unclear how this signal is transduced. The aim of this study is to investigate the different signaling pathways that have been implicated in NPM/ALK signaling to elucidate their role in the expression of C/EBPβ.
Materials and methods: To analyze the different signaling pathways induced by NPM-ALK, Ba/F3 cells were transfected with an NPM-ALK kinase-inhibitable construct (NPM-ALK-ATP-Abl). Imatinib was used to block NPM-ALK activity. Highly effective shRNA sequences (&gt;85% knockdown) were identified for AKT, mTOR, and Stat3 proteins using a specific lacZ reporter fusion assay in HEK-293T cells, and corroborated by Western blot analysis. Each of these shRNAs were cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Three ALK+ ALCL cell lines were analyzed (SUDHL-1, Karpas 299 and Ki-JK), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantitate the knockdown effect. These studies were supplemented with pharmacological inhibitors: rapamycin, MAPK inhibitors (U0126 and PD98059) and AKT inhibitor (Calbiochem). The effect of Stat3, AKT, mTOR and MAPK knockdown on proliferation and cell viability was analyzed by MTT assay and FACS analysis.
Results: Ba/F3 cells transfected with NPM-ALK-ATP-Abl construct resulted in induction of C/EBPβ expression and phosphorylation of Stat3, AKT and MAPK with no changes observed in mTOR phosphorylation. The opposite effect was observed when the NPM-ALK-ATP-Abl activity was inactivated with Imatinib. The infection rates of the specific shRNA constructs in the three ALK+ALCL cell lines were almost 100%. Downregulation of Stat3 in ALK+ALCL cells inhibited C/EBPβ at mRNA and protein level with impairment in cell proliferation and viability. In contrast, downregulation of AKT and mTOR showed no changes in C/EBPβ expression, whereas their downstream targets (rpS6 and 4E-BP1) phosphorylations were inactivated. These results were corroborated with rapamycin and AKT pharmacological inhibitory studies. MEK inhibitors (U0126 and PD98059) blocked the ERK1/2 phosphorylation reflected in growth retardation and its downstream target TSC2 phosphorylation without changing the expression of C/EBPβ. However, the phosphoThr-235 C/EBPβ was deactivated, confirming the importance of ERK1/2 in the phosphorylation and activation of C/EBPβ.
Conclusions: In this study, we demonstrated that the induction of C/EBPβ expression by NPM-ALK correlates with the phosphorylation of AKT, MAPK and Stat3. However, only the downregulation of Stat3 has influence on C/EBPβ mRNA and protein expression, whereas MAPK is important for the phosphorylation and modulation of CEBPβ function. The downregulation of C/EBPβ, as a consequence of Stat3 inhibition has an important effect on cell growth and survival.