CBX3/HP1γ promotes tumor proliferation and predicts poor survival in hepatocellular carcinoma

Expression and prognostic significance of CBX3/ HP1γ in various cancers

CBX3/HP1γ mRNA expression levels were examined in various cancers using the Oncomine (Figure 1A) and Human Protein Atlas (Figure 1B) databases. Most cancer tissues showed moderate to strong nuclear expression of CBX3/HP1γ. However, most lymphomas, prostate, and renal cancers displayed weak or negative staining. Bioinformatic analysis indicated that upregulated CBX3/HP1γ expression predicts poor prognosis in liver cancer (Figure 1C), breast cancer (Figure 1D), pancreatic cancer (Figure 1E), and renal cancer (Figure 1F) (Kaplan-Meier plot, log-rank test, all P values< 0.001).

CBX3/HP1γ expression is higher in liver cancer than in normal tissues

CBX3/HP1γ expression was compared in liver cancer and normal liver tissues in the following six datasets from the Oncomine and TCGA databases (Figure 2): Roessler Liver 2, Chen Liver, Roessler Liver, Guichard Liver, TCGA, and Wurmbach Liver. CBX3/HP1γ mRNA expression was elevated in liver cancer tissues compared to normal tissues. In the two Roessler datasets, CBX3/ HP1γ mRNA expression was 1.89-fold (p=7.91E-63) and 1.70-fold (p=2.12E-5) higher in HCC tissues [34]. Similarly, in the Chen Liver [35], Guichard Liver [36], Wurmbach Liver [36], and TCGA datasets, CBX3/HP1γ mRNA expression was increased 1.32- (p=1.85E-8), 1.05- (p=2.36E-9), 1.48- (p=6.23E-8), and 1.09-fold (p=3.93E-8), respectively, in HCC tissues.

Upregulation of CBX3/HP1γ is associated with poor prognosis in liver cancer

CBX3 expression in liver cancer and normal liver tissues was verified using the UALCAN database (Figure 3). The median transcript level in primary tumors was 60.5 per million compared to 29.6 per million in normal tissue (p=1.62e-12). We explored the prognostic significance of CBX3/HP1γ expression level in liver cancer using the Human Protein Atlas database (Figures 1C and 3B), the UALCAN web-portal (Figure 3C, log-rank test p<0.0001), and the Kaplan–Meier plotter survival analysis platforms (Figure 3D, HR=2.37 (1.67-3.36), log-rank test p=6.9E-7). High CBX3/HP1γ expression was associated with unfavorable prognosis in liver cancer.

Elevated CBX3/HP1γ expression in human HCC tissue specimens based on Western blot and qPCR analyses

Next, we compared CBX3/HP1γ protein and RNA expression levels in three pairs of matched HCC tumor and adjacent non-tumor tissue using Western blots and qPCR. Both CBX3/HP1γ protein and mRNA expression were elevated in HCC tumor tissues compared to matched adjacent nonneoplastic tissues (Figure 4A–4C).

IHC staining of CBX3/HP1γ in HCC tissue and its prognostic value in HCC patients

We performed IHC staining of HCC tissue microarrays containing samples from 354 HCC patients to evaluate the relationship between CBX3/HP1γ expression and HCC prognosis. CBX3/HP1γ protein expression was mainly located in the nucleus (Figure 4D). Based on our IHC scoring regime and the ROC curve cutoff value, patients were divided into low CBX3/HP1γ expression (negative or low expression in tumor cells; Figure 4E) and high CBX3/HP1γ expression (high expression in tumor cells; Figure 4D) groups. We then examined the relationship between CBX3/HP1γ expression levels and clinicopathological characteristics (Table 1). Among these, AFP > 20 μg/L, liver cirrhosis, tumor size > 5 cm, multiple tumors, vascular invasion, and tumor recurrence were associated with high CBX3/HP1γ expression (Chi square test P<0.05). In addition, CBX3 IHC scores were negatively correlated with overall survival (OS) times (Figure 4F).

To confirm the correlation between CBX3/HP1γ expression levels in tumor cells and HCC prognosis, we compared OS and recurrence-free survival (RFS) between the high and low CBX3/HP1γ expression groups. RFS was defined as the time from surgery to first recurrence of HCC or death without evidence of disease. Kaplan-Meier survival analysis revealed that patients in the high expression group had poorer OS and RFS times than those in the low expression group (Figure 4G and 4H).

Univariate and multivariate survival analyses were conducted to determine whether high CBX3/HP1γ expression in tumor cells was an independent prognostic factor for HCC. Univariate survival analysis revealed that CBX3/HP1γ expression, AFP, GGT, liver cirrhosis, tumor size, tumor number, vascular invasion, and tumor capsule were significantly associated with OS. Meanwhile, GGT, liver cirrhosis, tumor size, tumor number, vascular invasion, differentiation grade, and tumor capsule were significantly associated with RFS. Multivariate survival analysis was also performed to exclude confounding factors. Hazard ratios for OS and RFS are shown in Table 2. CBX3/HP1γ expression level (HR=1.375; 95% CI 1.080–1.928; P=0.045), liver cirrhosis (HR=1.537; 95% CI 1.065–2.218; P=0.022), tumor size (HR=2.214; 95% CI 1.419–3.455; P < 0.001), tumor number (HR=3.066; 95% CI 2.116–4.441; P < 0.001), and vascular invasion (HR=2.499; 95% CI 1.720–3.633; P < 0.001) were independent prognostic factors for OS in HCC patients. In addition, liver cirrhosis (HR=1.582; 95% CI 1.112–2.250; P=0.011), tumor size (HR=2.255; 95% CI 1.461–3.482; P < 0.001), tumor number (HR=2.952; 95% CI 2.038–4.276; P < 0.001), and tumor differentiation grade (HR=3.301; 95% CI 1.535–7.102; P=0.002) were independent prognostic factors for RFS in HCC patients in this Cox regression model (Table 3).

Stable overexpression of CBX3/HP1γ promotes growth in HCC cell lines

Because higher CBX3/HP1γ expression was associated with malignant clinicopathological features in HCC patients, we explored the potential biological function of CBX3/HP1γ in HCC tumorigenesis. The HCC cell lines SK-Hep-1 and SMMC-7721 were stably transfected with a CBX3/HP1γ expression plasmid (pEZ-Lv105-CBX3) or a control vector (pEZ-Lv105). Ectopic CBX3/HP1γ messenger RNA and protein expression in transfected cells was confirmed by quantitative real-time PCR and Western blot analyses, respectively (Figure 5A–5C). Functional CCK-8 and colony-forming assays were used to characterize proliferation ability in cells overexpressing CBX3/HP1γ. As shown in Figure 5D and 5E, cells overexpressing CBX3/HP1γ generated significantly more colonies than cells expressing only the vector in both the CCK-8 and colony-forming assays.

Molecular mechanisms underlying the effects of CBX3/HP1γ

The GeneMANIA and STRING web portals were used to predict interactions between CBX3/HP1γ and other genes (Figure 6A and 6B). The results showed that CBX3/HP1γ interacts with CBX1 and CBX5, which are members of the heterochromatin protein 1 family. The CircNet database was used to predict upstream circRNAs and microRNAs of CBX3/HP1γ (Figure 6C and 6D). These results indicated that CBX3 expression may be related to circ-CBX3.7 and microRNAs such as miR-30a.

Bioinformatics analysis

CBX3/HP1γ mRNA levels were examined in various cancers, including liver cancer, using the ONCOMINE database (http://www.oncomine.org) and The Cancer Genome Atlas database (TCGA, https://cancergenome.nih.gov/), which is a publicly accessible online database. The Human Protein Atlas database (https://proteinatlas.org/), Kaplan–Meier plotter survival analysis platforms (http://kmplot.com/), and UALCAN database (http://ualcan.path.uab.edu/) were employed to examine CBX3/HP1γ expression and survival in different cancers. Potential interactions between CBX3/HP1γ and different genes, microRNAs, and circRNAs were predicted using GeneMANIA (http://genemania.org/), STRING (http://string905.embl.de/), and CircNet (http://syslab5.nchu.edu.tw/CircNet/).

Patients and specimens

HCC tissue microarrays containing 354 samples from patients who underwent hepatic resection for HCC at Sun Yat-sen Cancer Center between 2005 and 2013 were examined. The inclusion criteria for patient enrollment were: an absence of anticancer therapies or distant metastasis prior to the surgery; a lack of concurrent autoimmune disease, human immunodeficiency virus, or syphilis; and the availability of follow-up data. Patients with Child-Pugh scores of B or C were excluded from the study. Histologic tumor differentiation grades were assigned according to the Edmondson-Steiner grading system. The BCLC staging system and the seventh edition of the International Union Against Cancer/American Joint Committee on Cancer TNM staging system were used for staging. The clinicopathological characteristics of the patients are summarized in Table 1. Freshly resected HCC specimens and adjacent non-neoplastic liver tissues were collected from patients who had undergone hepatectomy for curative treatment of HCC at the Sun Yat-sen Cancer Center. None of these patients received any neoadjuvant therapies before surgery, including radiotherapy or chemotherapy. This study was approved by the institutional review boards of the Sun Yat-sen Cancer Center. Written informed consent was obtained from all patients, including for the use of their liver specimens for research.

Immunohistochemistry

HCC tissue microarrays were cut into 3-μm sections for immunohistochemistry. Tissue sections were prepared for antigen retrieval using microwave treatment in citrate buffer (pH 6.0) and then incubated with a rabbit anti-CBX3 antibody (diluted 1:400 in PBS; Catalog number: 11650-2-AP, Wuhan Sanying Biotechnology, Wuhan, China) overnight at 4°C, followed by incubation with a secondary anti-rabbit HRP-conjugated antibody (Envision System, Catalog number: GK500711, Dako Cytomation, Glostrup, Denmark) at a concentration of 1:100 at 37°C for 30 min. Subsequently, the sections were washed with PBS, colorized with a diaminobenzidine (DAB) solution, and counterstained with hematoxylin. To minimize inter-observer variation, IHC staining was evaluated with a computerized image analysis platform that was constructed using the TMAJ Image application (http://tmaj.pathology.jhmi.edu) [7]. The cut-off point for low versus high expression was determined using the Youden index (sensitivity + specificity - 1) according to receiver operation characteristic (ROC) curves [38].

Follow-up

Follow-ups consisted of physical examination, tumor marker measurement, liver biochemistry and function, blood tests, abdominal ultrasonography, and contrast-enhanced CT and were conducted for all patients less than every 1-3 months for the first year after liver operations and every 6 months thereafter for more than 60 months after treatment. The overall survival (OS) time was defined as the interval between surgery and death or between surgery and the last follow-up for surviving patients. The recurrence-free survival time was defined as the interval between surgery and recurrence or between surgery and the last follow-up for patients without recurrence. The median follow-up was 63.5 months (range 2–155 months). Of the 354 patients examined during the follow-up period, 160 patients (45.2 %) died and 157 patients (44.6 %) did not experience tumor recurrence.

Cell lines and culture conditions

The human hepatocellular carcinoma (HCC) cell lines SK-Hep-1 and SMMC-7721 and the human embryonic kidney 293T (HEK 293T) cell line were obtained from the Liver Cancer Institute of Fudan University (Shanghai, China). All cell lines were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). The cells were incubated at 37°C in a humidified incubator supplied with 5% carbon dioxide.

Establishment of CBX3/HP1γ overexpression cells

A lentiviral CBX3/HP1γ overexpression vector was purchased from Hanbio Biotechnology Co., Ltd (Shanghai, China) and transfected into SK-Hep1 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells transfected with empty vector were used as controls. Puromycin was used to select stable clones.

Western blot analysis

Tissues and cells were lysed on ice in 50 mM Tris (pH 7.5), 150 mM NaCl, and 0.5% NP-40. Protein lysates were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membranes. After the membranes were blocked with 5% bovine serum albumin, they were incubated with primary antibody (CBX3 or GAPDH: Catalog number: 60004-1-Ig, Wuhan Sanying Biotechnology, Wuhan, China) at 4°C overnight. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 45 min. Protein signals were detected using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Bands were quantified using ImageJ (Ver. 1.52a; NIH, Bethesda, MD), normalized to GAPDH, and expression ratios were determined.

RNA isolation and quantitative real-time PCR

Total RNA was isolated from the tissue specimens and cell lines using TRIzol Reagent (Invitrogen Life Technologies, Shanghai, China). The isolated RNA (2 μg) was reverse-transcribed to cDNA using a SuperScript® III First-Strand Synthesis System (Invitrogen Life Technologies) according to the manufacturer’s instructions. For the qPCR assay, the cDNA was subjected to PCR amplification using SYBR Green (Toyobo, Kita-ku, Osaka, Japan) and a Roche LightCycler 480 System. GAPDH was used as an internal control. The primers used included the CBX3/HP1γ forward primer (5′-TAGATCGACGTGTA GTGAATGGG-3′), CBX3/HP1γ reverse primer (5′-TGT CTGTGGCACCAATTATTCTT-3′), GAPDH forward primer (5′- GTCTCCTCTGACTTCAACAGCG-3′), and GAPDH reverse primer (5′-ACCACCCTGTTGCTGTA GCCAA-3′).

Invitro cell growth assays

The proliferative activity of cultured cells was determined using Cell Counting Kit-8 (CCK8) assays (Promega, WI, USA); numbers of viable cells were evaluated after 2 h of incubation in medium containing CCK8. The conversion of the tetrazolium salt WST-8 to formazan was measured at 450 nm using a plate reader. For colony formation assays, 1,000 cells were seeded in each well of a 6-well plate and cultured with DMEM (Gibco) supplemented with 10% FBS (Gibco) for 7 d. The colonies were washed twice with phosphate-buffered saline (PBS), fixed in methanol for 15 min, and stained with crystal violet for 15 min at room temperature. After the excess stain was washed out, the number of colonies was counted. Colony formation efficiency was calculated as the ratio of the number of colonies formed to the total number of cells plated.

Statistical analyses

For continuous variables, the data are expressed as the mean ± standard error of the mean. The significance of differences between values was determined using the Student’s t-test. The chi-squared test was used to examine associations between CBX3/HP1γ expression and clinical pathological parameters. Patient survival curves were calculated using the Kaplan-Meier method and analyzed using the log-rank test. Prognostic factors were examined by univariate and multivariate analyses using the Cox proportional hazards model. All differences were deemed significant at P < 0.05. All statistical analyses were performed using SPSS software version 19.0 (SPSS, Chicago, IL).