C/EBPβ Is a Transcriptional Regulator of Wee1 at the G₂/M Phase of the Cell Cycle

Abstract

The CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that regulates cellular proliferation, differentiation, apoptosis and tumorigenesis. Although the pro-oncogenic roles of C/EBPβ have been implicated in various human cancers, how it contributes to tumorigenesis or tumor progression has not been determined. Immunohistochemistry with human non-small cell lung cancer (NSCLC) tissues revealed that higher levels of C/EBPβ protein were expressed compared to normal lung tissues. Knockdown of C/EBPβ by siRNA reduced the proliferative capacity of NSCLC cells by delaying the G₂/M transition in the cell cycle. In C/EBPβ-knockdown cells, a prolonged increase in phosphorylation of cyclin dependent kinase 1 at tyrosine 15 (Y15-pCDK1) was displayed with simultaneously increased Wee1 and decreased Cdc25B expression. Chromatin immunoprecipitation (ChIP) analysis showed that C/EBPβ bound to distal promoter regions of WEE1 and repressed WEE1 transcription through its interaction with histone deacetylase 2. Treatment of C/EBPβ-knockdown cells with a Wee1 inhibitor induced a decrease in Y15-pCDK1 and recovered cells from G₂/M arrest. In the xenograft tumors, the depletion of C/EBPβ significantly reduced tumor growth. Taken together, these results indicate that Wee1 is a novel transcription target of C/EBPβ that is required for the G₂/M phase of cell cycle progression, ultimately regulating proliferation of NSCLC cells.

Keywords: C/EBPβ; G2/M arrest; Wee1; Y15-pCDK1; cell cycle; lung cancer.

Figure 1

CCAAT/enhancer-binding protein β (C/EBPβ) expression in human lung cancer tissues. (A) Patients-derived lung cancer tissue arrays were examined for C/EBPβ expression using the immunoperoxidase method. Staining results were graded according to the intensity and proportion of positive area. Images were captured at a magnification of 200X by using the Aperio ImageScope software. Scale bars: 200 μm. (B) The histogram represents the percentage of the immunohistochemistry (IHC) score for C/EBPβ in 68 normal tissues, 95 primary, and 9 metastatic tumor tissues. The statistical significance was determined using the t-test, p < 0.05. (C) The association between C/EBPβ mRNA expression and overall survival of adenocarcinoma (whole dataset) and squamous cell carcinoma patients (GSE37745) was analyzed using the Kaplan–Meier Plotter. Hazard ratio (HR) significance was found with log-rank tests.

Figure 2

C/EBPβ promotes cell proliferation in various subtypes of non-small cell lung cancer (NSCLC) cell lines. (A) C/EBPβ protein levels in normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells, BEAS-2B and various NSCLC cell lines were determined by Western blot analysis. Aenocarcinoma, A549, NCI-H1975, NCI-H23, NCI-H1703, NCI-H522, A427, Calu-3, NCI-H358; adeno-squamous cell carcinoma, HCC2279; squamous cell carcinoma, HCC95, HCC1588; large cell carcinoma, H460, NCI-H1299; anaplastic carcinoma, Calu-6. (B) Live cells of A549 transfected with si-Negative Control (siNC), siC/EBPβ #1, or siC/EBPβ #2 were counted with trypan blue staining at indicated times after transfection. Data are presented as fold increase. (C) Doxycycline-inducible shC/EBPβ cells using A549 were generated and treated with or without doxycycline (100 ng/mL). Using the IncuCyte live cell imaging system, proliferation cells was monitored and quantified by the percentage of cell confluence. (D) The protein levels of C/EBPβ were detected by Western blotting to check C/EBPβ-knockdown in each cell line. (E) Cell number of lung cancer cell lines transfected with siNC or siC/EBPβ (#1 + #2) was counted using a Coulter counter at intervals of 24 h up to 120 h after siRNA transfection. K-RAS: mutant K-RAS/wild-type EGFR, EGFR: wild-type K-RAS/mutant EGFR, WT: wild-type K-RAS/wild-type EGFR. Data are presented as mean ± standard deviation (SD). The statistical significance was determined using t-tests, * p < 0.05, ** p < 0.01.

Figure 3

C/EBPβ-knockdown inhibits cell cycle progression. (A) A549 cells were transfectected with siNC or siC/EBPβ and incubated for 48 h. As a positive control for cell death, cells were treated with 20 M cisplatin for 48 h. Live cells were stained with green calcein-AM, while dead cells were stained with red ethidium homodimer-1 (EthD-1). Cell images were taken at a magnification of 100X using an Operetta High Content Screening (HCS) System. Scale bar: 200 μm. (B) A549 cells were transfected with control siRNA or C/EBPβ siRNA for 48 h. The cell cycle was analyzed by fluorescence-activated cell sorting (FACS) after DNA staining with propidium iodide (PI). M1: subG₀/G₁, M2: G₀/G₁, M3: S, and M4: G₂/M phase. Percentage of cells in each cell cycle phase is shown as a bar graph. (C) Whole cell lysates were prepared 48 h after transfection and the levels of the G₂/M cell cycle-related proteins in control or C/EBPβ-knockdown cells were analyzed by Western blotting. β-actin was used as a loading control. Data are presented as mean ± SD. Statistical significance was determined using the t-test, * p < 0.01.

Figure 4

C/EBPβ knockdown delays G₂/M-cell cycle transition. (A) A time course study of the cell cycle analysis was performed in control or C/EBPβ-knockdown A549 cells. Cells were released from thymidine double block-induced G₁/S synchronization; and 0, 2, 4, 6, 8, 10, 12, 14, 16, and 26 h after releasing, cells were collected and stained with PI to measure DNA content using FACS. (B) The data are expressed as the percentage of cells in the subG₀/G₁, G₀/G₁, S, and G₂/M phase at the indicated time points. (C) Whole cell lysates were prepared at the indicated times and Western blot analysis was performed for expression of proteins associated with the G₂/M transition (Y15-pCDK1, Wee1, Cdc25B and Cyclin B1). SE; short exposure, LE; long exposure. (D) Mitotic duration of control or C/EBPβ-knockdown A549 cells stained with NucBlue^® was monitored by an Operetta High Content Screening (HCS) System. Representative images of siNC and siC/EBPβ-transfected cells from time lapse series were shown. Images were acquired every 10 min. Data are presented as mean ± SD of mitotic duration in 30 cells in each group. Mitotic duration was measured from nuclear envelope breakdown (prometaphase) to anaphase onsets [52]. Statistical significance was determined using the t-test; n.s., not significant. Cell images were taken at a magnification of 200X using an Operetta High Content Screening (HCS) System. Scale bar: 20 μm.

Figure 5

C/EBPβ regulates Wee1 expression at the transcription levels and interacts with HDAC2. (A) Quantitative real-time RT-PCR (qRT-PCR) was used to determine Wee1 and Cdc25B mRNA levels relative to the control gene GAPDH in C/EBPβ-knockdown A549 cells. Data are presented as mean ± SD. (B) The position of the four predicted C/EBPβ binding sites in the WEE1 promoter are represented. C/EBPβ–ChIP on chip data and TFSEARCH based binding sites are indicated as a rectangle and an oval, respectively. The prediction of WEE1 promoter regions was based on NCBI accession number (NC_000011.10, GRCh37.p11) (C) The C/EBPβ-ChIP assay followed by qRT-PCR on putative C/EBPβ binding regions on the WEE1 promoter was performed to determine endogenous C/EBPβ occupancy at the specified region. The fold enrichment of C/EBPβ occupancy over GAPDH exon (negative control) is shown. Data are presented as mean ± SE. (D) A549 cell lysates were immunoprecipitated using anti-C/EBPβ antibodies. Immunocomplexes were analyzed by Western blot with either anti-HDAC1 or -HDAC2 antibodies. IgG was used as a negative control. (E) HDAC2-ChIP assay followed by qRT-PCR on putative C/EBPβ binding regions at the WEE1 promoter was performed. The fold enrichment of HDAC2 occupancy over GAPDH exon (negative control) is shown. Data are presented as mean ± SE. (F) The C/EBPβ-ChIP or HDAC2-ChIP assay followed by PCR on putative C/EBPβ binding regions at the WEE1 promoter was performed. The PCR products resolved on 2% agarose gel were visualized. (G) A549 cells were co-transfected with a WEE1 promoter-luciferase construct containing R2, or R3 along with C/EBPβ and/or HDAC2, as indicated, for 48 h, and then luciferase activities were measured. Data are expressed as relative luciferase activity/ug protein standardized by a control pGL3-promoter vector. Data are presented as mean ± SD. Statistical significance was determined using the t-test, * p < 0.05.

Figure 6

C/EBPβ knockdown cells treated with MK1775 recovered from the delay in the G₂/M phase. (A,B) A549 cells transfected with siNC or siC/EBPβ were treated with either DMSO or MK1775 4 h after being released from thymidine double block. Cells were harvested for cell cycle analysis at different time points after release, and DNA contents with PI staining were analyzed using FACS. (C) Expression of cell cycle-associated proteins was analyzed in DMSO- or MK1775-treated control cells and C/EBPβ-knockdown cells. Percentage of cells in each cell cycle phase is shown as a bar graph. SE; short exposure, LE; long exposure.

Figure 7

C/EBPβ-knockdown inhibits tumor growth. (A) A549 cells (5 × 10⁶) were implanted subcutaneously into athymic nude mice. When tumor size reached 60 to 80 mm³, siNC or siC/EBPβ were delivered into the tumors via electroporation once a week for seven weeks. Tumors were measured at the indicated time and tumor volume was calculated as described in Section 2. Photos from siNC- or siC/EBPβ-treated tumors are shown. Similar results were observed in three independent experiments. Data are presented as mean ± SD. The statistical significance was determined using the t-test, * p < 0.05, ** p < 0.01, significantly different from siC/EBPβ-treated tumor volume. (B) Immunohistochemical staining for C/EBPβ, Y15-pCDK1, Wee1, Cdc25B, Ki67, and cleaved caspase-3 (CC-3) was conducted with paraformaldehyde-fixed, paraffin-embedded xenograft tumors. Images were captured at a magnification of 400X by using the Aperio ImageScope software. Scale bars: 100 μm.

Figure 8

Schematic representation of the potential role of C/EBPβ in the G₂/M phase of cell cycle progression. (A) C/EBPβ represses WEE1 transcription by directly binding to WEE1 distal promoter regions and recruiting HDAC2. (B) Wee1 and Cdc25B are key regulators of phosphorylation of tyrosine 15 residue of CDK1, which blocks mitotic entry. C/EBPβ activates Cdc25B expression via unknown mechanism but inhibits Wee1 expression. In the absence of C/EBPβ, cells undergo G₂/M delay displaying increased CDK1 phsophorylation along with increased Wee1 and decreased Cdc25B levels.