Negative Regulation of C/EBPbeta1 by Sumoylation in Breast Cancer Cells

Sumoylation is a post-translational modification that is oftentimes deregulated in diseases such as cancer. Transcription factors are frequent targets of sumoylation and modification by SUMO can affect subcellular localization, transcriptional activity, and stability of the target protein. C/EBPbeta1 is one such transcription factor that is modified by SUMO-2/3. Non-sumoylated C/EBPbeta1, p52-C/EBPbeta1, is expressed in normal mammary epithelial cells but not breast cancer cell lines and plays a role in oncogene-induced senescence, a tumor suppressive mechanism. Although p52-C/EBPbeta1 is not observed via immunoblot in breast cancer cell lines, higher molecular weight bands are observed when breast cancer cell lines are subjected to immunoblot analysis with a C/EBPbeta1-specific antibody. We show that exogenously expressed C/EBPbeta1 is sumoylated in breast cancer cells, and that the higher molecular weight bands we observe in anti-C/EBPbeta1 immunoblots of breast cancer cell lines is sumoylated C/EBPbeta1. Phosphorylation oftentimes enhances sumoylation, and phosphorylation cascades are activated in breast cancer cells. We demonstrate that phosphorylation of C/EBPbeta1Thr235 by Erk-2 enhances sumoylation of C/EBPbeta1 in vitro. In addition, sumoylated C/EBPbeta1 is phosphorylated on Thr235 and mutation of Thr235 to alanine leads to a decrease in sumoylation of C/EBPbeta1. Finally, using a C/EBPbeta1-SUMO fusion protein we show that constitutive sumoylation of C/EBPbeta1 completely blocks its capability to induce senescence in WI38 fibroblasts expressing hTERT. Thus, sumolylation of C/EBPbeta1 in breast cancer cells may be a mechanism to circumvent oncogene-induced senescence.