Identification of Genes Which Play a Crucial Role in C/EBPβ Downstream Signalling in ALK⁺ ALCL Cell Lines.

ALK⁺ anaplastic large cell lymphomas (ALCL) overexpress C/EBPβ, as a consequence of NPM-ALK kinase activity. We recently reported C/EBPβ as a transcription regulator of NPM-ALK induced cellular proliferation. To identify the downstream targets of C/EBPβ that might be responsible for cell proliferation and survival, we performed gene expression profiling and pathway analyses after C/EBPβ gene silencing

C/EBPβ knockdown was done by lentiviral shRNA-transduction into two ALK⁺ ALCL cell lines with strong C/EBPβ expression – SUDHL1 and KiJK. At day three after infection, RNA was extracted and used for Gene Chip expression analysis (U133 Plus 2.0 arrays/ Affymetrix). Genes regulated in both cell lines were applied to Genomatix Bibliosphere Pathway analysis. Candidate genes were either strongly influenced by C/EBPβ knockdown, or had promoter binding sites for C/EBPβ, or showed remarkable pathway connections. The influence of C/EBPβ on these genes was validated by qRT-PCR and in part by Western blot.

Gene expression profiling analysis resulted in 167 genes being regulated in both cell lines, of which 26 genes were chosen for further analysis. Validation by qRT-PCR confirmed 23/26 genes. Pathway analysis revealed c-Jun, which is a member of the dimeric transcription factor AP-1, as a regulator of C/EBPβ expression. Silencing C/EBPβ led to a clear up-regulation of c-Jun mRNA. Western blot analysis demonstrated that C/EBPβ influenced not only the expression of c-Jun but also its phosphorylation on Ser63 and Ser73. In contrast to what has been reported, we found very low levels of c-Jun expression in ALK⁺ALCL cells lines and its expression correlated inversely with C/EBPβ mRNA levels. Although it has been shown that c-Jun regulates C/EBPβ expression directly, in ALK⁺ALCL the expression of C/EBPβ is clearly independent of c-Jun. Our data suggest that c-Jun up-regulation after C/EBPβ knockdown is a compensatory mechanism to maintain C/EBPβ expression. Additionally, of the 26 selected genes, Bibliosphere Analysis identified 12 genes, which might be transcriptionally regulated by C/EBPβ and are primary targets in C/EBPβ downstream signalling. Two of these genes are of particular interest. The anti-apoptotic protein BCL2A1 contains a promoter-binding site for C/EBPβ and has been shown previously to be both strongly regulated in ALK⁺ALCL and absolutely necessary for its transformation. The second is a DEAD box nucleolar RNA helicase protein involved in ribosomal RNA production and proliferation which we found to be strongly expressed in ALK⁺ALCL cell lines and primary cases.

C/EBPβ silencing in ALK⁺ALCL cell lines showed 1) an inverse correlation between c-Jun and C/EBPβ mRNA expression levels, 2) the expression of C/EBPβ in ALK⁺ALCL is independent of c-Jun, 3) genes transcriptionally regulated by C/EBPβ seem to be essential for proliferation and survival in ALK⁺ALCL.

No relevant conflicts of interest to declare.