Translational control of C/EBPβ isoform expression: a focus on regulation and function in breast cancer

The widely expressed C/EBPβ transcription factor regulates various cellular processes such as differentiation, proliferation, migration, senescence and apoptosis to maintain tissue homeostasis. Three protein isoforms (LAP1, LAP2 and LIP) are translated from the single‐exon CEBPB‐mRNA through the usage of alternative translation initiation sites. Synthesis of LIP depends on translation of a cis‐ regulatory uORF element in the CEBPB‐mRNA 5’‐leader sequence and subsequent translation re‐ initiation. LIP lacks the N‐terminal transactivation domains of LAP1/2 but contains an identical C‐ terminus including the DNA binding/dimerization domain. Therefore, LIP binds to the same genomic sites as LAP1/2 but inhibits target gene transcription due to its inability to recruit transcription co‐ activators. The LIP/LAP ratio therefore determines the transcriptional activity of C/EBPβ. Previous studies have shown LIP stimulates mammary epithelial proliferation and is highly expressed in triple negative breast cancer (TNBC) and correlates with poor prognosis. Also, mice deficient in C/EBPβ‐LIP display an increased health‐ and lifespan with reduced overall tumor incidence while overexpression of LIP promotes tumorigenesis in mice. Recent work from our lab established that LIP promotes cell migration and invasion in TNBC cells. LIP translation is mediated by both canonical and non‐canonical translation initiation factors and can be dynamically regulated in response to extracellular stimuli such as nutrient deprivation via the mTORC1 pathway. We find that the high LIP expression observed in TNBC cell lines and patient samples is refractory to mTORC1 inhibition. In search for the mechanisms behind mTORC1 insensitivity and the high levels of LIP expression in TNBC, C/EBPβ reporters were developed for use in a genome‐wide CRISPR/Cas9 screening approach. Furthermore, we identify the mTORC1 substrate S6K rather inhibits LIP translation in all models tested through a yet to be identified mechanism. In search of novel regulators of C/EBPβ translation, we find m6A modification of CEBPB‐ mRNA affects LIP/LAP expression ratios. Knockdown of m6A demethylase FTO reduces breast cancer growth and migration with a concomitant decrease in LIP expression. Our data support a role for FTO in regulating cell migration and epithelial‐mesenchymal transition (EMT), at least in part through modulation of LIP/LAP expression, while FTO deficiency induces increased sensitivity to E2F inhibition. We further find deregulated expression of LIP causes increased activity of the malate‐aspartate shuttle (MAS). Although this helps cells to proliferate under nutrient rich conditions, it induces sensitivity to glycolysis inhibition as the continued MAS activity profoundly reduces the cytosolic NADH/NAD+ ratio. In all, our data support a role for C/EBPβ isoforms in the regulation of breast cancer growth, migration and metabolism that warrants future research focusing on the therapeutic potential of CEBPB‐mRNA translation modulation. Finally, we establish a mouse model deficient in C/EBPα‐p30, the sister isoform of C/EBPβ‐LIP, by mutation of the C/EBPα‐uORF. Initial characterization of this model revealed isoform‐ specific C/EBPα functions in glucose metabolism, while future efforts will investigate the role of C/EBPα‐p30 in (hematopoietic) malignancy and health‐ and lifespan.