CUL4A facilitates hepatocarcinogenesis by promoting cell cycle progression and epithelial-mesenchymal transition

CUL4A, a member of the CULLIN family, functions as a scaffold protein for an E3 ubiquitin ligase. It was reported that the CUL4A gene showed amplification in some human primary hepatocellular carcinomas (HCC). However, the exact role of CUL4A in HCC remains unknown. Here, we aimed to investigate the expression and function of CUL4A in HCC development. Through immunohistochemistry study, we showed increased CUL4A expression in HCC tissues. Statistical analysis disclosed an inverse correlation between CUL4A expression and tumor differentiation grade, and patient survival, but a positive correlation with hepatocyte proliferation as well as lymphatic and venous invasion. CUL4A expression in HCC tissues was associated with HBeAg status in patients and upregulated by HBV in HCC cell lines. Further functional assay showed that CUL4A overexpression significantly promoted growth of H22 tumor homografts in BALB/c mice. Consistently, CUL4A knockdown inhibited the proliferation of established HCC cells, accompanied by S-phase reduction and Cyclin A and Cyclin B1 repression. Furthermore, CUL4A siRNA ameliorated the motility of HCC cell lines with altered expression of epithelial-mesenchymal transition (EMT)-associated molecules. Taken together, our findings indicate that CUL4A plays a pivotal role in HCC progression and may serve as a potential marker for clinical diagnosis and target for therapy.


Results
CUL4A is highly expressed in HCC tissues. Previous studies showed the amplification of CUL4A gene in HCC cell lines and clinical samples 5 . Here we examined CUL4A expression in normal liver tissues and HCC tissues by immunohistochemistry staining. Weak or intermediate immunoreactivity of CUL4A was detected in normal liver tissues (Fig. 1A). Similar CUL4A immunoreactivity was also observed in paratumor tissues (Fig. 1B, upper right panel). However, CUL4A expression was significantly increased in HCC tissues (Fig. 1B, lower left panel), when compared with paired adjacent non-tumor tissues (Fig. 1C). Moreover, high level of CUL4A was positively associated with tumor pathological grade (Fig. 1D,E). CUL4A expression in poorly-differentiated tumors was significantly higher than that in well-differentiated or moderately-differentiated tumor tissues (P = 0.0006, Table 1). In addition, increased CUL4A staining correlated with reduced overall survival times of patients (Fig. 1F). Consistently, levels of hepatocyte proliferation (Ki-67 as the marker) were significantly higher in CUL4A-positive HCC tissues (staining score: 5-12) than CUL4A-negative tissues (staining score: 0-4) (Fig. 1G). Moreover, the increase of CUL4A expression was more significant in tumors with diameters more than 5 cm and in tumors with lymphatic and venous invasion (Table 1). Collectively, our data demonstrate that increased CUL4A expression correlates with the progression of HCC and CUL4A might be involved in promoting tumor growth and metastasis.
HBV and its viral proteins upregulate CUL4A expression. Based on our data, CUL4A expression was increased in about 71% HCC tissues, which was significantly higher than the frequency of the CUL4A gene amplification in HCC tissues (3/51) reported before 5 . The difference suggested that there were other factors contributing to the upregulation of CUL4A in HCC tissues. It is well-known that HBV infection is a major etiological factor for HCC development 17 . In our study cohort, almost all patients were serum HBsAg positive. Considering that HBeAg could be an indicator of HBV replication 18 , we analyzed the correlation of serum HBeAg status in patients with CUL4A expression. As shown in Fig. 2A, serum HBeAg-positive patients had a higher CUL4A expression than HBeAg-negative patients. Moreover, both full-length HBV genome and HBV encoded proteins (HBx, preS2, and HBc) enhanced CUL4A mRNA expression in HCC cell lines (Fig. 2B,C). Consistently, the expression of CUL4A protein was also significantly upregulated by full-length HBV genome and viral proteins HBx and preS2 (Fig. 2D,E). Taken together, these data indicate that HBV infection contributes to the upregulation of CUL4A in HCC patients.

CUL4A promotes HCC growth both in vivo and in vitro.
To further investigate the role of CUL4A in HCC development, we measured the growth of H22 tumor homografts in BALB/c mice where CUL4A expression was modulated. After the tumor diameter reached 0.5 cm, pCMV-Tag2B or pCMV-CUL4A was injected intratumorally. Tumor growth curve showed that pCMV-CUL4A significantly promoted the growth of H22 tumor homografts over the period of the experiment (Fig. 3A,B). Consistently, pCMV-CUL4A injected tumors at the time of sacrifice weighed more than pCMV-Tag2B injected group The influence of CUL4A on HCC growth was further validated by measuring the growth of HCC cell lines in vitro. The HCC cell lines HepG2 and BEL7402, with high endogenous level of CUL4A expression, were transfected with two siRNAs specifically targeting CUL4A. The proliferation of both cell lines was significantly inhibited by CUL4A siRNA over a four-day period (Fig. 3E,G). The knockdown efficiency of CUL4A in both cell lines was confirmed by Western blot (Fig. 3F,H). Taken together, both in vivo and in vitro studies indicate that CUL4A promotes cell growth and support the idea that CUL4A functions as an oncogene in HCC development.

CUL4A knockdown correlates with S-phase reduction and Cyclin A and Cyclin B1 repression.
To explore the mechanisms for CUL4A promoting the proliferation of HCC cells, cell cycle profile in two HCC cell lines, where CUL4A expression was knocked down, was analyzed by flow cytometry. Transfection of CUL4A siRNA coincided with the decreased percentage of S phase cells in both HepG2 (Fig. 4A,C) and BEL7402 cells (Fig. 4B,D). In addition, the expression of potential cell cycle regulators was examined. Western blot results showed that CUL4A knockdown accompanied with the downregulated expression of Cyclin A and Cyclin B1 in both HepG2 (Fig. 4E) and BEL7402 cells (Fig. 4F). Collectively, these results indicate that CUL4A expression correlates with the deregulated cell cycle progression and the altered expression of cell cycle-related proteins in HCC cell lines.
CUL4A knockdown inhibited HCC cell migration and invasion by reversing EMT tendency. Our analysis with clinical samples suggested that increased CUL4A expression correlated with lymphatic and venous invasion of HCC tissues (Table 1). To further evaluate the role of CUL4A in controlling cell motility, Transwell assay combined with Matrigel analysis were performed in both HCC cell lines transfected with CUL4A siRNA. As shown in Fig. 5A,C, silencing CUL4A dramatically weakened the migratory and invasive capacity of HepG2 cells. Similar results were also obtained in BEL7402 cells (Fig. 5B,D). Thus, these results indicate that CUL4A enhances the migration and invasion of HCC cell lines.
Considering the importance of EMT in the progression of HCC 19 , we determined the expression pattern of epithelial and mesenchymal markers on HCC cell lines transfected with CUL4A-siRNAs or control NC-siRNA. Western blot results showed that silencing CUL4A increased the level of epithelial markers (E-Cadherin), but decreased the expression of mesenchymal markers (Vimentin) as well as EMT-promoting transcription factors including snail, slug and twist ( Fig. 5E,F). Also, we detected actin fiber formation in CUL4A knockdown or control cells by immunofluorescence. As shown in Fig. 5G, compared with control cells, CUL4A silencing BEL7402 and HepG2 cells showed reduced actin fiber formation. Taken together, these results support the idea that CUL4A promotes the metastatic property of HCC cells by inducing EMT.

Discussion
CUL4A was described as an oncogene in many kinds of tumors [6][7][8][9][10][11][12][13][14] . Amplification of CUL4A gene in several HCC cell lines and clinical HCC samples 5 indicated its potential involvement in the pathogenesis of HCC. However, the exact role of CUL4A in the development and progression of HCC remains unknown.
In the present study, we for the first time reported that CUL4A protein expression was significantly upregulated in HCC tissues. More importantly, aberrant expression of CUL4A was more pronounced in poorly-differentiated HCC tissues and tumors more than 5 cm in diameter, indicating that CUL4A participates in the progression of HCC. One important question is how CUL4A expression is upregulated in HCC. It is well-accepted that complicated genetic or epigenetic alterations in hepatocytes are involved in finely tuning the expression of oncogenes and tumor suppressor genes, which in turn transform the normal hepatocytes and lead to hepatocarconogenesis 4 . Although it was reported previously that CUL4A gene amplification occurred in several HCC cell lines and tumor tissues 5 , it could not fully explain the overexpression of CUL4A protein occurring in most of HCC cases, as indicated in our immunohistochemistry staining assay. Thus, there might be other factors contributing to the upregulation of CUL4A in HCC development. HBV infection is the most important cause of HCC worldwide. It is well-established that HBV and its encoded proteins greatly contribute to these processes by regulating the expression of multiple tumor-related genes, such as human telomerase reverse transcriptase (TERT), β -catenin, and Snail, etc 20 . In our study cohort, ~90% of HCC cases are HBsAg positive. Interestingly, the statistical analysis showed the positive correlation    between the presence of serum HBeAg and CUL4A expression in HCC tissues, indicating the regulatory role of HBV on CUL4A expression. Moreover, transfection of full-length HBV genome and viral gene fragment, HBx, HBc and preS2, had a significant induction of CUL4A expression. All these data suggest that HBV might be closely associated with the upregulation of CUL4A in HCC. However, the exact mechanisms for this role of HBV needs to be further investigated.
Consistent with upregulation of CUL4A in poorly-differentiated and large-size tumors, both in vitro and in vivo assay demonstrated that CUL4A had the ability to promote the proliferation of HCC cells. This raises another important question, how CUL4A promotes the growth of HCC cell lines? It is well-known that CUL4A induces cell cycle progression in normal cells by regulating the expression of cyclin-dependent kinase (CDK) inhibitors (CDIs), such as p21 CIP1/WAF1 , p27 KIP1 , and p16 INK4a 7 , which is important for the maintenance of normal cell proliferation and survival. However, in tumor cells, overexpression of CUL4A instead deregulates cell cycle and then results in the uncontrolled proliferation of malignant cells 21,22 . Here, we found that silencing CUL4A correlated with the reduced S-phase cells and downregulation of Cyclin A and Cyclin B1 (Fig. 4), while the expression of other cell cycle-related proteins remained unchanged (our unpublished data). Cyclin A, binding with CDK2, is responsible for controlling S phase progression and G2/M transition 23 . Cyclin B1 associates with CDK1 and promotes the process of mitotic cell division 24 . The deregulated expression of Cyclin A and Cyclin B1 was involved in reinforcing the malignant growth of HCC 25,26 . Detailed mechanisms for CUL4A modulation on Cyclin A and Cyclin B1 expression requires further study.
The correlation between high CUL4A expression and lymphatic and venous invasions suggests a potential role of CUL4A in metastasis of HCC tumors. The in vitro transwell assay also supports our hypothesis. Furthermore, Western blot results showed that CUL4A knockdown upregulated the expression of epithelial marker (E-cadherin), but downregulated the steady-state level of mesenchymal marker (Vimentin), and EMT-associated transcription factors. Also, immunofluorescence study indicated that CUL4A silencing reduced the actin fiber formation. EMT has been demonstrated to play an important role in the migration, invasiveness, metastasis and chemoresistance and has been highlighted as potential therapeutic target of HCC 27,28 . Our data indicate that CUL4A might accelerate the progression of HCC by promoting EMT tendency, although the exact mechanisms by which CUL4A affects EMT progression needs to be further investigation.
In conclusion, our study indicates that CUL4A controls cell proliferation and Cyclin A and Cyclin B1 are upregulated. These data provide new evidence for the tumor-promoting roles of CUL4A in liver cancer. Also, we identify the roles of CUL4A in the migration and invasion of HCC cell lines, which might be associated with EMT progression. Further studies are required to understand the detailed mechanisms by which CUL4A promotes cell cycle and EMT progression in HCC.

Materials and Methods
Clinical HCC samples. Fifty-seven tumor tissues and the corresponding adjacent non-tumor tissues were collected from patients with primary HCC who underwent surgical resection between Jan. 2012 and Mar. 2013 at Shandong University Qilu Hospital and Shandong Provincial Hospital. Cell differentiation-based HCC tumor grading was determined by the experienced pathologists according to the criteria described by Edmondson and Steiner 29 . HBV antigens in serum of patients were determined by ELISA. Patients with evidence of HCV or HIV infection, consumed excessive alcohol or received chemotherapy prior to surgery were excluded. Written informed consent was obtained from all patients, and the study was approved by the Shandong University Medical Ethics Committee in accordance with the Declaration of Helsinki. Immunohistochemistry staining. Immunohistochemistry was performed according to standard protocols using the following antibodies: anti-CUL4A (ab34897, Abcam) and anti-Ki-67 (ab15580; Abcam). Ten fields of ~1000 cells from each tumor and non-tumor section was independently counted by three pathologists. The level of CUL4A expression was reported according to the German semi-quantitative scoring system 30,31 . Briefly, CUL4A expression in each sample was scored according to staining intensity (no staining = 0; weak staining = 1; moderate staining = 2; strong staining = 3) and the number of stained cells (0% = 0; 1-25% = 1; 26-50% = 2; 51-75% = 3; 76-100% = 4). The final scores of immunoreactive were determined by multiplying the staining intensity by the number of stained cells, with minimum and maximum scores of 0 and 12, respectively 32 . Different staining grade was ranked according to the final staining scores: 0, total score = 1; 1+ , total score = 1-4; 2+ , total score = 5-8; 3+ , total score = 9-12. The levels of hepatocyte proliferation were shown by the staining intensity of Ki-67, which was determined as described for CUL4A staining.
Cell lines, plasmids and siRNAs. The human HCC cell lines HepG2 and BEL7402 were purchased from the Shanghai Cell Collection, Chinese Academy of Sciences. HepG2 cells were cultured in DMEM, and BEL7402 cells were maintained in RPMI1640. All culture media were supplements with 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 U/ml streptomycin (Invitrogen, Beijing, China). Murine liver cancer cell line H22 was maintained in our laboratory and was passaged weekly in ascites fluid of BALB/C mice 33  The plasmid pCMV-CUL4A was constructed by cloning mouse full-length CUL4A gene fragment into pCMV-Tag2B as described previously 34 . The plasmids containing 1.1-fold genome of HBV, HBx, HBc, or preS2 gene fragment were constructed on the backbone of the mammalian expression vector pcDNA3 as described previously [35][36][37] .
In vivo tumor homograft assay. Six to eight week-old, male BALB/c mice were purchased from the Animal center of Shandong University (Jinan, China) and housed in ventilated microisolator cages in a pathogen-free facility. Individual mice were implanted subcutaneously with 2 × 10 5 H22 murine liver cancer cells suspended in 100 μ l phosphate buffer solution. When the diameter of tumors reached 5 mm, the mice were randomly divided into two groups (6 mice for each group) and injected intratumorally with pCMV-Tag2B or pCMV-CUL4A (20 ug in 100 ul saline) every three days for a total 3 injections. The tumor sizes in each mouse were monitored using a vernier caliper every other day until three days after the last injection. Then, the mice were sacrificed and the tumors were isolated and weighed. CUL4A expression in tumors was performed by RT-PCR. All experiment protocols were carried out in accordance with the guidelines, which were approved by the Shandong University Animal Care Committee.
Cell proliferation assay. The impact of CUL4A expression on the proliferation of HCC cells was determined by cell proliferation assay as described previously 38 . Briefly, BEL7402 or HepG2 cells at 10 4 cells/well were cultured in 96-well plates overnight and transfected with CUL4A-siRNA or NC-siRNA using LipofectamineTM 2000 (Invitrogen), followed by incubation for four days. The proliferation of HCC was determined longitudinally using the CCK-8 kit (Beyotime, Shanghai, China) and measuring the absorbance at A450 in a microreader (Bio-Rad, Tokyo, Japan). CUL4A expression was quantified by Western blot.
Cell cycle analysis. BEL7402 or HepG2 cells were collected 48 hrs after transfection with CUL4A-siRNA or NC-siRNA, stained with propidium iodide (PI, Sigma) and then assayed using a Beckman Coulter Flow Cytometer (Fullerton).
In vitro invasion and migration assay. The cell migration assay was performed by using Transwell (Corning Life Sciences, Acton, MA). Around 3 × 10 4 transfected cells (HepG2 or BEL7402) suspended in serum-free media were seeded on the upper side of the transwell chamber, which was transferred to a 24-well containing full culture medium containing 10% FBS. Twelve hours after incubation at 37 °C, cells on the upper side of the chamber were removed by wiping the top of the membrane with cotton swabs and the cells migrated to the lower side of the transwell membrane were fixed and stained in 0.1% crystal violet. Cells were counted from ~10 microscope fields and the mean value was shown. For cell invasion assay, transfected cells were seeded into matrigel (BD Biosciences, San Jose, CA) -coated transwell chamber and were incubated for 24 hours before staining. Each experiment was performed in replicate inserts and the mean value expressed from three independent experiments. Immunofluorescence staining and microscopy. Cells were seeded on cover slips, fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) and then stained with Tetramethylrhodamine (TRITC)-conjugated phalloidin (Sigma-Aldrich, Louis, USA) For nuclear staining, cells were stained with 4' ,6-diamidino-2-phenylindole (DAPI) (Beyotime). Images were taken using a confocal laser microscope (Carl Zeiss, LSM780, Oberkochen, Germany). Western Blot. BEL7402 or HepG2 cells, transfected with CUL4B-siRNA or NC-siRNA for 48 h, were harvested and total cell lysates were prepared, as previously described 35 . The cell lysates (20 μ g/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% fat-free dry milk, the membranes were incubated with antibodies against CUL4A (ab34897, Abcam), Cyclin A (4656, Cell Signal Technology), Cyclin B1 (ab32053, Abcam), Twist (ab50581, Abcam), Slug (9585, Cell Signaling), Snail (3879,Cell Signaling), Vimentin (BS1491, Bioworld), E-cadherin (3195, Cell Signaling), and β -actin (A5316, Sigma, St. Louis, USA), respectively. The bound antibodies were detected with HRP-conjugated second antibodies and visualized using Western Lightning Chemilluminescence Reagent (Amersham Biosciences, Piscataway, USA).
Semi-quantitative and real-time RT-PCR analysis. BEL7402 cells were transfected with pcD-NA3-HBV1.1, pcDNA3-HBx, pcDNA3-HBc, pcDNA3-preS2, or pcDNA3. Total RNA from cells transfected for 48 h or H22 tumor tissues, was extracted using TRIzol (TaKaRa, Tokyo, Japan), according to the manufacturers' protocol. The total RNA was reversely transcribed into cDNA, and the relative levels of CUL4A mRNA transcripts to the internal control β -actin were determined by semi-quantitative or real-time PCR using the specific primers.
Scientific RepoRts | 5:17006 | DOI: 10.1038/srep17006 Statistical Analysis. GraphPad Prism (GraphPad Software, San Diego, CA) was used for data analysis. The differences between groups were determined by the Student t-test or Mann-Whitney U test. The statistical correlation between the clinical parameters of HCC and the CUL4A staining levels in tissue sections was analyzed by the Chi-square test. Survival differences were analyzed using the log-rank test. In these analyses, p values < 0.05 were considered to be statistically significant.