Int J Clin Exp Pathol 2018;11(10):4908-4916 www.ijcep.com /ISSN:1936-2625/IJCEP0080284 Original Article CREB5 promotes cell proliferation and correlates with poor prognosis in hepatocellular carcinoma Jian Wu1*, Shu-Tong Wang1*, Zi-Jian Zhang2, Qi Zhou1, Bao-Gang Peng1 1Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, PR China; 2Department of General Surgery, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen, Guangdong Province, PR China. *Equal contributors. Received May 25, 2018; Accepted September 7, 2018; Epub October 1, 2018; Published October 15, 2018 Abstract: CAMP responsive element binding protein 5 (CREB5) has been reported to be overexpressed in several types of human cancers and has crucial roles in regulating cell growth, proliferation, differentiation, and the cell cycle. However, the expression and function of CREB5 in hepatocellular carcinoma (HCC) remains unclear. The purpose of this study was to investigate the role of CREB5 in HCC, and its prognostic significance. We measured the expression of CREB5 in 91 specimens of paraffin-embedded HCC tissue by immunohistochemistry and performed a clinicopathological analysis. Gain of function and loss of function assays were used to evaluate the effect of CREB5 on cell proliferation in vitro. We found that up-regulation of CREB5 was associated with a poor prognosis, and CREB5 status was an independent prognostic factor. The overexpression of CREB5 increased the proliferation of SMMC- 7721 cells, but the knockdown of CREB5 had the opposite effect. The results indicate that CREB5 may be useful when determining a treatment strategy for patients with HCC. Keywords: CREB5, HCC, proliferation, prognosis Introduction tal cancer, lung cancer, and ovarian cancer [10- 12]. However, the expression and function of Hepatocellular carcinoma (HCC) is the fifth CREB5 in HCC remains unknown. Thus, the pur- most common cancer in the world and repre- pose of this study was to examine the expres- sents the third leading cause of cancer mortal- sion of CREB5 in HCC and determine its prog- ity worldwide with a 30% to 40% 5-year postop- nostic significance. erative survival rate [1-3]. The high mortality of HCC is mainly related to HCC metastasis, which Materials and methods is a major cause of tumor recurrence after HCC patient samples tumor resection [4-7]. However, the molecular mechanism of HCC metastasis remains largely Ninety-one patients with a median follow-up unknown. Therefore, it is critical to identify the time of 18 months with HCC who underwent mechanisms underlying HCC migration and hepatectomy at the First Affiliated Hospital of invasion in order to develop novel therapeutic Sun Yat-sen University were included in the strategies. study. All patients were diagnosed with primary liver cancer, and none had received preopera- CAMP responsive element binding protein 5 tive radiotherapy or chemotherapy. The study (CREB5) belongs to the CRE (cAMP response was carried out under the approval of the element)-binding protein family. The protein Committees for Ethical Review of Research specifically binds to CRE as a homodimer or involving Human Subjects of the First Affiliated heterodimer with c-Jun or CRE-BP1 to function Hospital of Sun Yat-sen University (Guangzhou, as a CRE-dependent transactivator [8, 9]. China). All patients, or their guardians or care- Previous studies suggested that CREB5 might givers, provided written informed consent for play a key role in the tumorigenesis of colorec- the use of clinical specimens in medical CREB5 promotes HCC Table 1. Relationship of CREB5 expression with ing to the proportion of positively stained tumor pathological parameters of HCC cells and the intensity of staining. Tumor cell Low CREB5 High CREB5 proportion was scored as follows: 0 (no positive Variables P Group Group tumor cells), 1 (< 35% positive tumor cells), 2 Age (yrs) (35-70% positive tumor cells), and 3 (> 70% ≤ 50 35 12 0.625 positive tumor cells). Staining intensity was graded according to the following criteria: 0 (no > 50 35 9 staining), 1 (weak staining = light yellow), 2 Sex (moderate staining = yellow brown), and 3 Male 60 20 446 (strong staining = brown). The staining index Female 10 1 was calculated as the product of the staining HBsAg intensity score and the proportion of positive Positive 58 19 0.508 tumor cells. Using this method of assessment, Negative 12 2 we evaluated CREB5 expression by determin- Tumor size (cm) ing the staining index, with scores of 0, 1, 2, 3, ≤ 10 cm 57 12 0.039 4, 6, or 9. An optimal cutoff value was identi- > 10 cm 13 9 fied: a staining index score of > 5 was used to Tumor number define tumors with high CREB5 expression, and Solitary 57 14 0.227 a staining index score of < 5 was used to define low CREB5 expression. Multiple 13 7 Vascular invasion HCC cell line Yes 9 4 0.488 No 61 17 The HCC cell line SMMC-7721 was obtained Encapsulation from the Cell Bank of the Chinese Academy of Intact 46 15 0.793 Medical Sciences (Shanghai, China). The cells Non-intact 24 6 were maintained in high-glucose Dulbecco’s Edmonson modified Eagle’s media (DMEM) (Gibco, Au- stralia) supplemented with 10% fetal bovine I-II 51 17 0.573 serum (Gibco, Australia). III-IV 19 4 AFP (μg/l) Plasmid constructs and siRNA transfection ≤ 400 40 13 0.803 > 400 30 8 Full-length CREB5 cDNA was amplified and TNM stage cloned into the pReciever M68 expression vec- I-II 59 10 0.001 tor (FulenGen, Guangzhou, China). The expres- III 11 11 sion plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s in- research. Of the 91 patients (80 men and 11 structions. women; mean age, 51 years; range, 21 to 73 years), 70 were serum hepatitis B virus (HBV)- Oligonucleotide siRNA duplex was synthesized positive, and 53 had an elevated alpha-fetopro- by Shanghai Gene Pharma (Shanghai, China). tein level (AFP; ≥ 200 ng/mL) (Table 1). The siRNA sequence of CREB5 was: 5’-GG- UGGAAAUGGACUGGCUTT-3’. The transfection Immunocytochemistry of siRNA in the HCC cells was carried out with Lipofectamine 2000 (Invitrogen), according to Paraffin-embedded sections of HCC tissue or the manufacturer’s instructions. adjacent non-cancerous liver tissue were cut and dehydrated in xylene, followed by micro- Cell cycle analysis wave antigen retrieval. The sections were incu- bated with an anti-ELMO3 antibody and then To evaluate the cell cycle, 1×106 cells were har- analyzed using the Evision/HRP Kit (Dako, CA, vested and washed in PBS, then fixed in 75% USA). The degree of immunostaining was alcohol for 30 min at 4°C. After washing in cold reviewed and scored by two observers accord- PBS three times, the cells were resuspended in 4909 Int J Clin Exp Pathol 2018;11(10):4908-4916 CREB5 promotes HCC min. The cells were stained with crystal violet, and images were collected by a video camera. Western blot analysis Tissues or harvested cultured cells were homogenized in a lysis buffer (50 mmol/L Hepes pH 7.5, 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10 mmol/L NaF, 10 mmol/L Na4P2O7, 1 mmol/L Na3VO5, 1 mmol/L phenyl- methylsulfonyl fluoride, 10 μg/ ml leupeptin and 20 μg/ml aprotinin). Protein was quanti- fied by the Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of protein were sepa- rated on SDS-polyacrylamide gels and transferred onto ni- trocellulose membranes (Am- ersham Biosciences, Pisca- taway, NJ). After blocking in 5% skim milk for 1 h at room temperature, membranes we- re incubated with a primary antibody at 4°C overnight, fol- lowed by a horseradish peroxi- dase-conjugated secondary an- Figure 1. Representative immunohistochemical staining of CREB5 in tumor tibody, and analysis by a che- tissues. The micrographs showed weak (A), medium (B), and strong (C) ex- miluminescence assay (Am- pression of CREB5 in liver cancer tissues. (Magnification: left panel 100×, right panel 400×). ersham Biosciences, Pisca- taway, NJ). Quantification of the Western blot data were 1 ml of PBS solution with 40 ug of propidium performed by measuring the intensity of the iodide (Sigma) and 100 ug of RNase A (Sigma) hybridization signals using and image analysis for 30 min at 37°C. Samples were then ana- program (Fluor-ChemTM 8900, Alpha Inotech). lyzed for their DNA content by FACSCalibur (Becton Dickinson, Mountain View, CA). The Statistical analysis proliferative index (PIx), a measure of the num- ber of cells in a tumor that are dividing (prolifer- The differences between the two groups were ating), was calculated with the following formu- examined using Student’s t test. A value of P < la: PIx = (S+G2M)/(G0/G1+S+G2M)]×100%. 0.05 was considered statistically significant. Data were expressed as the mean ± standard Colony formation assays deviation from at least three experiments. Comparisons between groups were performed For colony formation assays, 500 cells were with two-tailed paired Student’s t test. The chi- plated onto 6-well plates and incubated at square test was used to analyze the relation- 37°C. When the cells had grown to form visible ship between the CREB5 expression and the colonies, the colonies were washed once with clinicopathological features. Bivariate correla- PBS and fixed with 4% paraformaldehyde for 20 tions between variables were determined by 4910 Int J Clin Exp Pathol 2018;11(10):4908-4916 CREB5 promotes HCC Figure 2. Kaplan-Meier survival curves of hepatocellular carcinoma (HCC) patients based on CREB5 expression. HCC patients with high CREB5 expression had a significantly shorter (A) overall survival (P = 0.004) and (B) disease- free survival (P = 0.017) than those with low CREB5 expression.