Abstract
The transcription factor C/EBPβ is a master regulator of mammary gland development and tissue remodelling during lactation. The CEBPB-mRNA is translated into three distinct protein isoforms named C/EBPβ-LAP1, -LAP2 and -LIP that are functionally different. The smaller isoform LIP lacks the N-terminal transactivation domains and is considered to act as an inhibitor of the transactivating LAP1/2 isoforms by competitive binding for the same DNA recognition sequences. Aberrantly high expression of LIP is associated with mammary epithelial proliferation and is found in grade III, estrogen receptor (ER) and progesterone (PR) receptor-negative human breast cancer. Here, we show that reverting the high LIP/LAP ratios in triple-negative breast cancer (TNBC) cell lines into low LIP/LAP ratios by overexpression of LAP reduces migration and matrix invasion of these TNBC cells. In addition, in untransformed MCF10A human mammary epithelial cells overexpression of LIP stimulates migration. Knockout of CEBPB in TNBC cells where LIP expression prevails, resulted in strongly reduced migration that was accompanied by a downregulation of genes involved in cell migration, extracellular matrix production and cytoskeletal remodelling, many of which are epithelial to mesenchymal transition (EMT) marker genes. Together, this study suggests that the LIP/LAP ratio is involved in regulating breast cancer cell migration and invasion. This study together with studies from others shows that understanding the functions the C/EBPβ-isoforms in breast cancer development may reveal new avenues of treatment.
Highlights
The CCAAT/enhancer-binding protein family of basic region leucine-zipper transcription factors consists of six members that are widely expressed and have functions involved in cell proliferation, differentiation, metabolism, and senescence[1,2]
Using CRISPR/Cas[9] mediated knockout of CEBPB and genome-wide RNA-sequencing we reveal that expression of genes associated with epithelial to mesenchymal transition (EMT) and the extracellular matrix (ECM) partially depend on LIP and/or LAP expression
We analysed C/EBPβ expression in the triple-negative breast cancer (TNBC) (ER−, PR−, HER2−) derived cell lines BT-20, MDA-MB-231, BT-549, HCC1806, MDAMB-468, Hs 578T and CAL-120, the luminal A derived (ER+, PR+, HER2−) cell lines MCF-7, T-47D and ZR-75-1, the HER2-positive derived cell line (ER−, PR−, HER2+) SK-BR-3, and the luminal B derived (ER+, PR+, HER2+) cell lines BT-474, MDA-MB-361 and ZR-75-3025 as well as in the untransformed mammary epithelial cell line MCF10A cultured in media supplemented with EGF that is known to stimulate LIP expression through CUGBP1 activation[26]
Summary
The CCAAT/enhancer-binding protein family of basic region leucine-zipper (bZIP) transcription factors consists of six members (designated α to ζ) that are widely expressed and have functions involved in cell proliferation, differentiation, metabolism, and senescence[1,2]. A high LIP/LAP expression ratio was linked to metastatic breast cancer samples that were defective in the TGFβ-induced cytostatic response, whereas a low LIP/LAP ratio was associated with an intact growth-inhibitory TGFβ response. Since evasion of the TGFβ cytostatic response is a critical step during breast cancer development towards a more migratory and invasive metastatic phenotype[19] these data suggest that C/EBPβ isoform deregulation (towards a higher LIP/LAP ratio) is crucially involved in promoting breast cancer metastasis[20]. To study the downstream transcriptional effects of the high LIP/ LAP isoform ratio, we analysed the transcriptional changes upon CRISPR/Cas[9] mediated knockout (ko) of CEBPB in the TNBC cell line BT-20 by genome-wide RNA sequencing (RNA-seq)
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