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

Abstract

Abstract 1943Poster Board I-966 Introduction: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 Materials and Methods: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. Results: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. Conclusions: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. Disclosures:No relevant conflicts of interest to declare.