C/EBPβ expression in ALK-positive anaplastic large cell lymphomas is required for cell proliferation and is induced by the STAT3 signaling pathway

Abstract

Background: Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma is characterized by the t(2;5) chromosomal translocation, resulting in the expression of a fusion protein formed of nucleophosmin (NPM) and ALK. Recently, we reported the abnormal expression of the transcription factor CCAAT/enhancer binding protein-beta (C/EBPbeta) in ALK-positive anaplastic large cell lymphomas, and demonstrated its dependence on NPM-ALK activity.

Design and methods: In this study, the role of C/EBPbeta in proliferation and survival of ALK-positive anaplastic large cell lymphomas was investigated, as well as the mechanism of its expression and activity. Highly effective short hairpin RNA sequences and/or pharmacological inhibitors were used to abrogate the expression or activity of C/EBPbeta, signal transducer and activator of transcription 3 (STAT3), AKT, extracellular signal-related kinase 1/2 (ERK1/2) and mammalian target of rapamycin (mTOR).

Results: Interference with C/EBPbeta expression resulted in a dramatic decrease in cell proliferation in ALK-positive anaplastic large cell lymphomas, with a mild induction of apoptosis after 6 days. Down-regulation of STAT3 resulted in a marked decrease in C/EBPbeta mRNA and protein levels with impairment in cell proliferation and viability, underscoring the important role of these two proteins in ALK-mediated oncogenesis. Additionally, we demonstrated that reduction of ERK1/2 activity led to C/EBPbeta Thr(235) dephosphorylation and moderate growth retardation. The AKT/mTOR signaling pathway did not have any influence on C/EBPbeta expression or C/EBPbeta phosphorylation.

Conclusions: These findings reveal the convergence of STAT3 and ERK1/2 signaling pathways activated by NPM-ALK in mediating the regulation of C/EBPbeta expression, a transcription factor central to NPM-ALK transformation.

Figure 1.

Influence of C/EBPβ shRNA-mediated gene silencing on the expression of the different C/EBPβ isoforms and on cell growth in ALK⁺ALCL cells. (A) FACS analysis of transduced SUDHL-1 cells with C/EBPβ shRNA and controls 3 days after infection. The percentage of GFP-positive cells represents the infected cells. (B) Western blot analysis of C/EBPβ knockdown three days after infection. The five specific C/EBPβ bands detected are labeled with numbers. The higher molecular weight band corresponds to LAP (46 kDa) and LAP* (48 kDa), and the lower to LIP (21 kDa). Three minor bands were observed at 39 kDa, 36 kDa and 30 kDa, possibly proteolytic breakdown products of the major LAP isoform. Each lane contained 30 μg protein extract. Tubulin was used as a loading control. (C) Proliferation curves of the controls and the C/EBPβ shRNA infected SUDHL-1 cells are depicted up to 6 days after infection. Error bars indicate SD (n=3). (D) Cell cycle distribution of the SUDHL-1 infected cells 3 days after infection with C/EBPβ-shRNA. The differences between the control and the C/EBPβ-shRNA-infected cells are given in percentages on the right side. (E) Annexin V staining of the controls and the C/EBPβ shRNA-infected SUDHL-1 cells 3 and 6 days after infection.

Figure 2.

ALK1, C/EBPβ, pmTOR, pSTAT3, pERK1/2 and pAKT expression in ALK⁺ ALCL and Ba/F3 cells. (A) The cell lines Ki-JK, SR 786, Karpas 299 and SUDHL-1 represent ALCL cell lines with the t(2;5) translocation. The five specific C/EBPβ reactive bands detected are labeled with numbers. (B) Western blot analysis of Ba/F3 cells with and without IL3, and after transfection with the NPM-ALK-ATP-Abl construct untreated or treated with 5 μM imatinib for 24 h. The NPM-ALK-ATP-Abl construct produces a positive protein band of 85 kDa, 5 kDa larger than the protein band detected in the SUDHL-1 cell line used as a control. Each lane contained 30 μg protein extract. Tubulin was used as a loading control.

Figure 3.

Influence of STAT3 shRNA-mediated gene silencing on the expression of C/EBPβ and on cell growth in ALK⁺ALCL. (A) FACS analysis of transduced SUDHL-1 cells with STAT3 shRNA and controls 3 days after infection. The percentage of GFP-positive cells represents the infected cells. (B) Western blot analysis of STAT3 and C/EBPβ in the transduced SUDHL-1 cells 3 days after infection. Each lane contained 30 μg protein extract. Tubulin was used as a loading control. (C) Quantitative RT-PCR analysis of STAT3 mRNA (dark boxes) and C/EBPβ mRNA (light gray boxes) in the transduced SUDHL-1 cells 3 days after infection. Values were normalized to TBP and data were analyzed according to the 2^-ΔΔCT method. Results are depicted as mRNA concentration relative to SUDHL-1 non-infected cells (SUDHL-1-c). Error bars indicate SD (n=2). (D) Proliferation curves of the controls and the STAT3 shRNA-infected SUDHL-1 cells are depicted up to 5 days after infection. Error bars indicate SD (n=3).

Figure 4.

Influence of MEK inactivation on C/EBPβ expression, C/EBPβ phosphorylation, and cell growth. (A) Western blot analysis of the protein extractions from SUDHL-1 cells after treatment with MEK inhibitor (U0126), 2 h and 24 h after inhibition. Each lane contained 30 μg protein extract. Tubulin was used as a loading control. (B) Proliferation assay of the control and the SUDHL-1 cells 2 h and 24 h after treatment with MEK inhibitor (U0126). Error bars indicate SD (n=3).

Figure 5.

Influence of shRNA-mediated mTOR and AKT inhibition on C/EBPβ expression and C/EBPβ phosphorylation. (A) The level of the knockdown effect for the mTOR protein and downstream targets (p-rpS6, p-4E-BP1) was analyzed by western blot from the SUDHL-1 cell extracts prepared 3 days after infection. Phospho-TSC2 and phospho-eIF2α were used as a control for specificity. (B) Quantitative RT-PCR analysis of mTOR mRNA (dark boxes) and C/EBPβ mRNA (light gray boxes) in the transduced SUDHL-1 cells 3 days after infection. Values were normalized to TBP and data were analyzed according to the 2^-ΔΔCT method. Results are depicted as mRNA concentration relative to SUDHL-1 non-infected cells (SUDHL-1-c). Error bars indicate SD (n=2). (C) The level of the knockdown effect for the AKT protein and phospho- C/EBPβ was analyzed by western blot from the SUDHL-1 cell extracts prepared 3 days after infection with corresponding controls and AKT shRNA. Phospho-STAT3 and phospho-mTOR were used as a control for specificity.