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

Sumoylation of C/EBPbeta1 in breast cancer cells.

<p>a. Cell lysates were prepared and were run on an 8% SDS-PAGE in the following order: lane 1 MCF10A, lane 2 MDA231, lane 3 MDA468, lane 4 BT-474, lane 5 SK-BR3, lane 6 MDA435 and lane 7 T47D. Immunoblot analysis was performed with a C/EBPbeta1-specific antibody raised to the first 21 amino acids unique to C/EBPbeta1 (Abcam 18F8). The bottom immunoblot was performed as a loading control for GAPDH. Bars indicate the mobility's of standard molecular weight markers, in kilo-Daltons (kDa), in all figures. b. MDA231 breast cancer cells were infected with T7-C/EBPbeta1-IRES-eGFP-LZRS three times and sorted by FACs using GFP as a marker. Immunoprecipitations of confluent 100 mm dishes were performed with uninfected MDA231s (lanes 1 and 3) or T7-C/EBPbeta1-MDA231 cells (lanes 2 and 4) using T7 antibody beads. The left is an immunoblot with an anti-SUMO-2/3 antibody and the immunoblot on the right is with an anti-C/EBPbeta antibody. Sumoylated C/EBPbeta1 is indicated and the parent p52 C/EBPbeta1 is indicated by the arrow. c. Immunoprecipitations of MDA468 cells were performed with protein A agarose beads cross-linked to a C/EBPbeta1-specific antibody (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025205#pone.0025205-Eaton1" target="_blank">[7]</a>). Lanes 3, 6, and 9 are the immunoprecipitations whereas lanes 1, 4, and 7 are negative control beads only and lanes 2, 5 and 8 are negative control non-crosslinked beads incubated with MDA468 extract. The left immunoblot is performed with an anti-SUMO-2/3 antibody, the middle immunoblot with a C-terminal C/EBPbeta antibody (Abcam 47A1) and the right hand immunoblot with a C/EBPbeta1-specifc antibody (Abcam 18F8). Arrows indicate sumoylated C/EBPbeta1. (231 = MDA231, beta1 = C/EBPbeta1, su = sumoylated).</p

Phosphorylation of C/EBPbeta1 by Erk-2 enhances sumoylation <i>in vitro</i> and sumoylated C/EBPbeta1 is phosphorylated on Thr235.

<p>a. Immunoblot analysis of purified rat C/EBPbeta1 (Lap1) (lanes 1 and 3) and C/EBPbeta1 incubated with purified, active Erk-2 (lanes 2 and 4). The immunoblot on the left is with the anti-T7 tag antibody and on the right is with the anti-phosphoThr235 C/EBPbeta antibody. Rat C/EBPbeta1 migrates faster via SDS-PAGE because it is smaller in size than human C/EBPbeta1. b. Immunoblot analysis with the anti-T7 tag antibody. Lane 1 is purified rat C/EBPbeta1, lane 2 is C/EBPbeta1 incubated with purified E1 SUMO activating enzyme, purified E2 SUMO conjugating enzyme, and purified SUMO-3 peptide, and lane 3 is C/EBPbeta1 with Erk-2, E1, E2 and SUMO-3. Arrows indicate C/EBPbeta1 and sumoylated C/EBPbeta1. c. Cos-7 cells were untransfected (lanes 1, 3, and 5) or transfected with T7-C/EBPbeta1-pcDNA3.1 and HA-SUMO-2-pcDNA3 (lanes 2, 4 and 6). All samples were immunoprecipiated with T7 antibody beads. Immunoblot analysis was performed with the anti-phosphoThr235 C/EBPbeta antibody (left), anti-HA tag (middle), and anti-C/EBPbeta antibody (right). Arrows indicate sumoylated T7-C/EBPbeta1 and p52-T7-C/EBPbeta1. (beta1 = C/EBPbeta1).</p

Mutation of Thr235 to alanine decreases sumoylation of C/EBPbeta1.

<p>a. Cos-7 cells were untransfected (lanes 1, 4, and 7), transfected with T7-C/EBPbeta1-pcDNA3.1 and HA-SUMO-2-pcDNA3 (lanes 2, 5 and 8), or transfected with T7-C/EBPbeta1T235A-pcDNA3.1 and HA-SUMO-2-pcDNA3 (lanes 3, 6, and 9). All samples were immunoprecipitated with T7 antibody beads. Immunoblot analysis was performed with the anti-phosphoThr235 C/EBPbeta antibody (left), anti-HA tag (middle), and anti-C/EBPbeta antibody (right). Arrows indicate sumoylated T7-C/EBPbeta1 and p52-T7-C/EBPbeta1. b. Immunoblot analysis using the anti-T7 tag antibody of cell lysates from Cos-7 (lane 1), Cos-7 cells transfected with T7-C/EBPbeta1-pcDNA3.1 and HA-SUMO-2-pcDNA3 (lane 2), and Cos-7 transfected with T7-C/EBPbeta1T235A-pcDNA3.1 and HA-SUMO-2-pcDNA3 (lane 3). Arrows indicate p52-T7-C/EBPbeta1 and sumoylated T7-C/EBPbeta1. The relative amount of protein in the parent T7-C/EBPbeta1 band and the 75 kDa sumoylated T7-C/EBPbeta1 band was measured using the LI-COR Odyssey system. It was determined that there is 3.25 times more sumoylated 75 kDa T7-C/EBPbeta1 as there is 75 kDa T7-C/EBPbeta1T235A. This was calculated relative to the p52-T7-C/EBPbeta1 and p52-T7-C/EBPbeta1T235A bands. This was repeated three times with a standard deviation of +/−0.26. (beta1 = C/EBPbeta1, su-beta1 = sumoylated C/EBPbeta1).</p

Sumoylated C/EBPbeta1 does not induce senescence.

<p>A. Equal cell numbers of the indicated cell lines were plated in 60 mm dishes and stained for senescence associated beta-galactosidase as per manufacturer's instructions (Cell Signaling Technology). Representative photomicrographs imaged with a light microscope are shown. B. Quantitative comparison of senescence associated beta-galactosidase positive cells. The experiment was repeated four times with equal cell numbers of the indicated cell lines ranging from 50,000–250,000 cells/60 mm dish. The density of plating did not affect the outcome. For each experiment the average number of beta-galactosidase positive (blue) cells in 10 fields was computed. Error bars indicated standard deviation of the mean. C. Whole cell extracts were prepared from the indicated WI-38hTert cells and analyzed by immunoblotting (bottom panel) or immunoprecipitated with T7 antibody beads (Novagen, upper panel) followed by immunoblotting. Immunoblot analysis was performed with N-terminal C/EBPbeta antibody developed in our lab and described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025205#pone.0025205-Eaton1" target="_blank">[7]</a>.</p

Symptom assessment in early psychosis: The use of well-established rating scales in clinical high-risk and recent-onset populations

Symptom assessment in early psychosis research typically relies on scales validated in chronic schizophrenia samples. Our goal was to inform investigators who are selecting symptom scales for early psychosis research. We described measure characteristics, baseline scores, and scale inter-relationships in clinical high-risk (CHR) and recent-onset psychotic disorder (RO) samples using the Positive and Negative Syndrome Scale, Brief Psychiatric Rating Scale, Scale for the Assessment of Positive Symptoms, and Scale for the Assessment of Negative Symptoms; for the CHR group only, we included the Scale of Prodromal Symptoms. For investigators selecting symptom measures in intervention or longitudinal studies, we also examined the relationship of symptom scales with psychosocial functioning. In both samples, symptom subscales in the same domain, across measures, were moderately to highly intercorrelated. Within all measures, positive symptoms were not correlated with negative symptoms, but disorganized symptoms overlapped with both positive and negative symptoms. Functioning was significantly related to negative and disorganized, but not positive, symptoms in both samples on most measures. Findings suggest strong overlap in symptom severity ratings among the most common scales. In recent-onset samples, each has strengths and weaknesses. In CHR samples, they appear to add little information above and beyond the SOPS