CR6-interacting factor 1 inhibits invasiveness by suppressing TGF-β-mediated epithelial-mesenchymal transition in hepatocellular carcinoma

CR6-interacting factor 1 (CRIF1) regulates cell cycle progression and the DNA damage response. Here, we show that CRIF1 expression is decreased in hepatocellular carcinoma (HCC) tissues and positively correlates with patients’ survival. In vitro, down-regulation of CRIF1 promotes HCC cell proliferation and invasiveness, while over-expression has the opposite effect. in vivo, CRIF1 knockdown enhances growth of HCC xenografts. Analysis of mRNA microarrays showed that CRIF1 knockdown activates genes involved in TGF-β RI/Smad2/3 signaling, leading to epithelial-mesenchymal transition (EMT) and increased matrix metalloproteinase-3 (MMP3) expression. However, cell invasion and EMT are abrogated in HCC cells treated with SB525334, a specific TGF-β RI inhibitor, which indicates the inhibitory effect of CRIF1 on HCC tumor growth is mediated by TGF-β signaling. These results demonstrate that CRIF1 benefits patient survival by inhibiting HCC cell invasiveness through suppression of TGF-β-mediated EMT and MMP3 expression. This suggests CRIF1 may serve as a novel target for inhibiting HCC metastasis.


INTRODUCTION
Deaths resulting from hepatocellular carcinoma (HCC) rank third in all malignancy-related mortality [1]. In China, liver cancer is the most commonly diagnosed cancer and the leading cause of cancer death in men under the age of 60 years [2]. Surgical resection and liver transplantation are the best options for HCC treatment. However, majority of HCC patients are not eligible for surgical operation due to metastasis, which is also a major obstacle to cure.
Systemic dissection of the molecular mechanisms underlying metastatic progression of HCC is necessary for the development of new diagnostic and therapeutic strategies to prevent and treat metastases. Epithelial-tomesenchymal transition (EMT) is a key event in metastasis [3,4]. CR6-interacting factor 1 (CRIF1), also known as Gadd45gip1 or PRG6, regulates cell cycle by inhibiting G1 to S phase progression, and binds to Gadd45 family [5], which plays an important role in genomic stability and regulation of the cell cycle. However, the precise role of CRIF1 in HCC has not been investigated. In this study, we evaluated the expression of CRIF1 in HCC tissues, and investigated its function in regulating HCC cell proliferation, in vivo tumor growth, EMT, and HCC metastasis.

CRIF1 expression is decreased in HCC patients, and correlates with patients' survival
First, we analyzed CRIF1 expression in human HCC tissues by immunohistochemistry (IHC). 38 out of 109 HCC tissues (34.9%) had high CRIF1 levels (staining score>6), while 66 of the 109 para-tumor tissues (60.6%) had high CRIF1 levels (chi-square test, P<0.001). CRIF1 was downregulated in HCC tissue ( Figure 1A, 1B, 1C, 1D and 1E). Pearson correlation analysis revealed that the CRIF1 protein level was tightly related to tumor differentiation (P = 0.049) and diameter (P = 0.047) ( Table 1). Univariate analysis demonstrated that tumor diameter, differentiation, and CRIF1 expression were of statistical significance. As shown in Figure 1F, Decreased CRIF1 expression in HCC tissues was associated with poor post-operative survival (P=0.018). The overall three-year survival rates for the low CRIF1 and high CRIF1 groups were 25.8% and 48.8% respectively (P=0.023).
CRIF1 suppression promotes proliferation of HCC Huh-7 and SK-HEP-1 cells CRIF1 knockdown promoted cell proliferation and invasive potency, while restoration of CRIF1 had the opposite effect (Figures 2A, 2B and 2C). Flow cytometry revealed that CRIF1 knockdown decreased the number of G1 phase cells and increased the number of G2 and S phase cells ( Figure 2D). In accordance, proteins controlling G1/S transition, including cyclin D1, cyclin   E1, CDK4, and CDK6 were significantly upregulated after CRIF1 knockdown ( Figure 2E).

CRIF1 inhibits HCC tumorigenicity in mice
To evaluate the role of CRIF1 in promoting HCC tumorigenicity in vivo, mice were subcutaneously injected with HCC cells with CRIF1 knockdown (CRIF1-KD) or normal control (NC) cells. As shown in Figure  3, four weeks after injection, the CRIF1-KD group had significantly larger tumors (1.115 +/-0.209 cm, n=8) than the CRIF1-NC group (0.496 +/-0.196 cm, n=8, P<0.001). Xenograft tumors in the CRIF1-KD group had elevated percentage of Ki-67 positive cells compared to the control group ( Figures 3C-3F).

Gene expression microarray
A total of 527 differentially expressed genes (fold change>2, p < 0.05) were identified between SK-HEP-1 cells transfected with siRNA-CRIF1 or siRNA-NC. The data were further analyzed with an online database (http://david.abcc.ncifcrf.gov/). The gene ontology analysis ( Figure 4A) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis ( Figure 4B) indicated involvement of genes and signaling pathways involved in the regulation of cell death, apoptosis, adhesion, and migration. Among them, MMP3 exhibited the most significant change. Knockdown of CRIF1 in HCC cells increased the mRNA expression of MMP3, Smad6, CDK6, and several other genes, while restoration of CRIF1 decreased the expression of these genes ( Figures  4C and 4D).
Since the activation of TGF-β signaling causes EMT and tumor metastasis, we investigated the impact of CRIF1 suppression on EMT in HCC cells. As shown in Figure  4E, we found a decrease of epithelial marker E-cadherin and elevation of mesenchymal markers N-cadherin and vimentin in CRIF1-suppresed cells. Snail, which binds to E-cadherin promoter region to repress transcription, was also upregulated. Meanwhile, tight junction-associated proteins claudin and ZO-1 were downregulated. Double immunofluorescent staining of E-cadherin and N-cadherin confirmed that CRIF1 knockdown promoted EMT ( Figure  5). When treated with TGF-β receptor I specific inhibitor SB525334, CRIF1 knockdown-activated TGF-β signaling, invasion, and EMT were abrogated ( Figure 6), suggesting that CRIF1 suppression promotes invasion and EMT in Huh-7 and SK-HEP-1 cells through TGF-β/Smad signaling.

DISCUSSION
Metastases account for the great majority of cancerassociated deaths, yet this complex process remains the least understood aspect of cancer biology [6]. Unraveling the mechanism of metastasis will open a new avenue to cancer cure. CRIF1 inhibits cell cycle, and functions as an essential STAT3 coactivator [7]. CRIF1 induces cell cycle arrest in the G1 phase by inhibiting the kinase activity of Cdk2-cyclin complexes, Cdk1-cyclin B1and histone H1 [8,9]. However, the role of CRIF1 in HCC has not been investigated.
Our data indicate that CRIF1 inhibits HCC cell proliferation, decreases the percentage of G1 phase cells, and inhibits in vivo tumorigenicity of HCC xenografts. In addition, our results are the first to demonstrate that CRIF1 inhibits HCC cell invasiveness, since CRIF1 knockdown increased HCC cells invasive potency, while restoring CRIF1 expression decreased their invasive potency.
Microarray data have indicated that several important genes including MMP3, PTEN, Smad6, and CDK6 are regulated by CRIF1 [10][11][12]. Among them, MMP3, a member of the matrix metalloproteinase (MMP) family, which is involved in the breakdown of extracellular matrix in processes, such as tissue remodeling and metastasis [13], had the largest fold change. The observed changes in MMP3 protein levels following CRIF1 knockdown or overexpression, may represent one of the mechanisms of how CRIF1 regulates invasiveness of HCC cells. TGF-β signaling, which plays pivotal roles in tumor metastasis [14], was among the most altered signaling pathways following CRIF1 knockdown. CRIF knockdown increased Smad2, Smad3, p-Smad2 and p-Smad3 expression, suggesting activation of the TGF-β signaling. Expression of TGF-β receptor I was increased after CRIF1 knockdown, implying that TGF-β receptor I plays a driving role in CRIF1-regulated TGF-β signaling.
Together, our results reveal a novel function of CRIF1 in regulating EMT and MMP3 expression in HCC cells, and suggest that CRIF1 may be an ideal target for inhibiting HCC metastasis.

Clinical sample preparation
This study was approved by the Ethical Committee of the First Affiliated Hospital of Zhejiang University School of Medicine, and conducted in accordance with the ethical principles of the Declaration of Helsinki. All clinical samples used in this study were obtained from patients after informed consents. A total of 109 paired specimens of HCC were randomly enrolled in this study from HCC patients undergoing hepatectomy from 2010 to 2012 at the First Affiliated Hospital of Zhejiang University School of Medicine.

Reagents and cell culture
The human HCC cell lines Huh-7 and SK-HEP-1 were purchased from Chinese Academy of Sciences Shanghai cell bank and cultured in complete growth medium, as recommended by the manufacturer. Cells were maintained at 37°C in a humidified incubator with 5% CO2. The TGF-β RI specific inhibitor SB525334 was purchased from Sellleck (Selleck Chemicals, Houston, TX, USA), and used at a concentration of 1 μM.

Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from tissues or cells with Trizol reagent (Invitrogen, USA). qRT-PCR was carried out using the master SYBR Green kit (Takara Bio, Japan) according to the manufacturer's instruction by ABI Prism 7500 fast sequence detection system (applied Biosystem, USA). All reactions were performed in triplicates, and analyzed by using the 2 -∆∆ Ct method. The results were normalized to mRNA expression of GAPDH, which was used as internal control.

Western blotting
Total proteins were extracted from tissues and cells.

CRIF1 siRNA interference, and recombinant plasmid and lentivirus infection
siRNA-CRIF1 and siRNA-NC (negative control) were synthesized by Invitrogen. The siRNA sequence for CRIF1 was ACACCUAGUAGGCUGCGUGCCUGUC. Hu-7 and SK-Hep-1cells were cultured in 6-well plates in MEM. When cells were about 30% confluent, they were transfected with siRNA-CRIF1 or siRNA-NC using transfection reagent (Lipofectamine 2000, Invitrogen, Carlsbad, CA). The CRIF1-recombinant and NC vectors were constructed by GeneChem. Stable CRIF1 knockdown (CRIF1-KD) and its negative control (CRIF1-NC) cell lines were obtained by infection of lentivirus targeting CRIF1 and the negative control. The efficiency of transfections was validated by qRT-PCR and western blotting. Fluorescence of CRIF1-KD and CRIF1-NC cells was observed after transfection and puromycin screening (Supplementary Figure 1). www.impactjournals.com/oncotarget

Cell proliferation assay
Cell proliferation was measured using the Cell Counting Kit-8 (Dojindo, Japan) assay. Briefly, pretreated HCC cells (transfected with siRNA or GV219) were placed in 96-well plates (normally 4000 cells per well) and cultured for indicated times. The absorbance was measured at 450 nm using Absorbance Reader (BIO-TEK ELX800); the experiments were repeated 3 times.

Tumor invasion assay
Polycarbonate membranes Transwell (Corning, USA) and Matrigel (BD, USA) were used for the migration assay. After different treatments, a total of 5×10 4 Hu-7 and SK-Hep-1cells were suspended in 200 μL of serum-free medium and seeded into the upper chamber, while 600 μL of MEM containing 10% FBS was added to the lower chamber. After incubation at 37°C, the cells on the upper membrane were removed with cotton swabs. The membranes were fixed in paraformaldehyde solution and stained with 0.5% crystal violet. Cells on the lower surface of the membrane were counted in randomly selected fields with microscope (100×). The experiment was independently repeated three times.

Xenograft tumorigenicity assay
All animal experiments were performed according to the Zhejiang University Animal Care Facility and National Institutes of Health guidelines.
Six-week-old BALB/c male nude mice (average weight 21 g) were randomly divided into two groups (8 mice each). Pretreated SK-Hep-1cells (4×10 6 cells in 0.1ml PBS) were injected subcutaneously into the nude mice. Tumor volumes were measured after mice were sacrificed.

Cell cycle and apoptosis assays
For cell cycle analysis, cells were harvested and fixed in ice-cold 75% ethyl alcohol at 4° C overnight. After incubation with a DNA PREP kit solution (Beckman Coulter, Fullerton, CA) in the dark for 30 min, cell apoptosis was detected by FACS. Data were analyzed using ModFit LT3.1 software. Apoptosis was detected in HCC cells not expressing GFP (Green Fluorescent Protein) using an FITC Annexin V Apoptosis Detection Kit II (BD, San Jose, CA), and in cells expressing GFP using an Annexin V Apoptosis Detection Kit APC (eBioscience, SanDiego, CA). Cells were analyzed using a flow cytometer (LSR II; BD Bioscience). All experiments were conducted in triplicates.

Statistical analysis
All values are expressed as mean ± standard deviation. Student's t-test was applied to evaluate statistical significance. The variable was converted to a logarithm if it did not conform to a normal distribution. Χ 2 test was used to detect the difference between categorical variables. The endpoint for follow-up was patient death. Overall survival rates were calculated using the Kaplan-Meier method. Log-rank tests were performed for univariate analysis. P-values less than 0.05 were considered statistically significant. All statistical analyses were performed using SPSS 11.0software (SPSS, Chicago, IL).

ACKNOWLEDGMENTS
We thank Jili Cao, Yuhui Liu, and Chao Wang for their technical help. We also thank Haijun Guo for his assistance in preparation of patient clinical information.

CONFLICTS OF INTEREST
No potential conflicts of interest were disclosed.