Treatment Of Hepatocellular Carcinoma With C/EBPα And Its Mechanism Investigation

[Background and Objective]Hepatocellular carcinoma (HCC) is the fifth common type of cancer worldwide but it is the third leading cause of cancer death. The high mortality rate of HCC is due to asymptomatic feature of HCC progression and unresponsiveness to therapy. Although substantial progress in the diagnosis and treatment of HCC has been made in the past decades, the prognosis for HCC in the advance stages is still poor which urgently call for new therapeutic methods.Hepatocyte nuclear factors (HNFs) family which includes a series of liver-enriched transcription factors (LETFs):HNF1, HNF3, HNF4, HNF6, C/EBP. It is highly express in well differentiated hepatic cells and regulates the expression of many important functional genes at the transcriptional level to promote hepatic differentiation, maintains biological function of hepatic cells and also plays an important role in the hepatic development. Previous studies indicated that the expression of HNFs was associated with the genesis and development of HCC. We have previously shown that forced re-expression of HNF4a can not only repress liver cirrhosis through reversal of hepatic cell EMT but prevent liver cancer progress by repressing wnt/β-cantanin signaling pathway. Additionally, we demonstrated that both HNFla and HNF4a exerted significant anti-tumor effect by inducing the differentiation of hepatoma cells into more mature phenotype both in vitro and in vivo.C/EBPa, a liver-enriched transcription factor, belongs to the basic leucine zipper superfamily of transcription factors. This protein is involved in the regulation of several biological processes in liver including energy metabolism, ammonia and bilirubin detoxification, acute phase response, tumor biology and cytochrome P450gene expression. Several studies have shown that C/EBPa may acts as a tumor suppressor in HCC. However, a recent study manifested that C/EBPa was up-regulated in a subset of HCCs by epigenetic regulatory mechanisms. Downregulation of C/EBPa resulted in the reduction of HCC tumorigenesis associated with a decrease in cyclin A, CDK4and E2F. These conflicting results suggest that the role of C/EBPa in HCC remain to be elucidated.Based on the central role of C/EBPa in hepatic development and cellular differentiation, herein, we detected the expressions of C/EBPa among the normal human hepatocytes, various hepatoma cells, the human HCC tissue and the tissue adjacent to the carcinoma. In addition, a replication-deficient adenovirus carrying C/EBPa (AdC/EBPa) was constructed with AdEasyTM system. The effect of AdC/EBPa on the expression of some liver function-related genes, proliferation, cell cycle and tumorigenesis of hepatoma cells were detected respectively. Furthermore, we determined the therapeutic effect of AdC/EBPa on different experimental HCC models of mice via intratumoral injection or the tail vein infusion. Finally, we reveal the mechanism of the C/EBPa suppression liver cancer growth by through Notchl/Hesl/p27cascades.[Methods]1. Expression of C/EBPa in hepatoma cell lines and human HCC tissuesThe expressions of C/EBPa in human mature hepatocytes, hepatoma cell lines were compared through Real-Time RT-PCR. With the same method, we also compared the C/EBPa expression in20human HCC tissues and adjacent tissues. And C/EBPa protein expression was measured by immunohistochemistry in6human HCC tissues and adjacent tissues.2. Construction of recombinant replication-deficient adenovirusThe C/EBPa cDNA was generated through restriction enzyme digestion from pcDNA3.1-C/EBPa. The shuttle plasmids-pAdTrack-CMV-C/EBPa was constructed by ligation. The linearized shuttle plasmids were co-transformed into coli bacillus BJ5183with backbone vector AdEasy-1to establish the recombinant adenoviral plasmids-pAdC/EBPa by homologous recombination.3. The effect of C/EBPa on the biological characteristics of hepatoma cells(1) Expression of C/EBPa mRNA and protein in hepatoma cells after AdC/EBPa infection After exogenous C/EBPa gene was delivered into the hepatoma cell lines Huh7real-time RT-PCR and western blot, and location of C/EBPa was detected by immunofluorescence.(2) Effect on expression of liver-specific genes in hepatoma cell lines after C/EBPa gene introductionExpression of liver-specific genes in hepatoma cell lines was detected by Real-Time RT-PCR at the third day of post-infected by the adenoviruses AdC/EBPa and AdGFP. Expression of liver-specific genes in Huh7cells was detected by real-time RT-PCR at the third day of post-transfected with siC/EBPa or siNC.(3) CCK-8method was used to detect the cell proliferation.(4) Influence on cell cycle in hepatoma cells by C/EBPa. Cell cycle arrest of the cells was analyzed by flow cytometry.(5) Colony formation ability of hepatoma cells after C/EBPa gene introduction.To evaluate colony formation ability of virus-infected hepatoma cells, the hepatoma cells were infected with Adenovirus.4×103infected cells were seeded onto10-cm culture dishes24h later and grew in culture medium for2weeks. Colonies were fixed with4%paraformaldehyde and visualized via crystal violet staining.(6) Cell Proliferation Assay for human cancer cells isolated from two independent samples (LIX006and LIXO10) and TAFs isolated from two independent samples (L100908B and L100825).(7) Tumorigenicity of hepatoma Huh7cellsTo detect the effect of C/EBPa on the tumorigenicity of hepatoma cells, four to six week old male BALB/c nude mice were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences and housed under standard conditions. To detect the effect of C/EBPa on the tumor formation ability of Huh7cells,4X106cells infected with AdC/EBPa or control virus AdGFP in200μl of serum-free medium were inoculated subcutaneously into the left and right armpits of each BALB/c nude mouse (8mice), respectively. Tumor size was measured with calipers, and volumes of tumors were determined using the following formula: Volume=width2×length×0.5. Mice were monitored for6weeks until the mice began to die for excessive tumor load. 4. Antitumor effect in vivoTo examine the antitumor effect of C/EBPa in vivo, subcutaneous HCC model was established in BALB/c nude mice. A total of4×106Huh7cells were inoculated into BALB/c nude mice subcutaneously. Intratumoral injection of2×109pfu AdC/EBPa or control virus AdGFP was conducted three times a week up to2weeks when the volumes of tumors reached about350mm3. Intratumor injection of AdC/EBPa were carried out three times a week up to2weeks. Mice were sacrificed after final treatment.To establish a liver xenograft model,4×106Huh7cells in a total volume of200μl of a serum-free medium were slowly injected into the liver. Intravenous delivery of2×109pfu adenoviruses via tail vein were carried out twice a week up to3weeks. Mice were sacrificed after final treatment and livers were removed for histologican and histopathological analysis.5. The effect of C/EBPa on Notch1signaling pathway.(1) Western blot and immunocytochemistry was used to detected the Notchl protein expression after infected by C/EBPa.(2) To determine whether C/EBPa inhibited Notchl activation affects transcriptionally regulated Notchl downstream target genes, we measured the change in RNA levels of c-Myc, cyclin D1, Hesl by real-time PCR. We further confirm our result by western blot.(3) To identify whether down-regulated Hesl by C/EBPa overexpression exerted biological effects on the growth of Huh7cells, CCK-8analysis of cell proliferation was examined among transfection with or without HES1followed infected with AdC/EBPa or AdGFP.(4) We next determined whether Notch signaling is required for C/EBPa regulation of Hesl expression. Huh7cells were transfected with or without NICD following infected with AdC/EBPa or AdGFP as control.(5) Sections from formalin fixed orthotopic liver tumor tissues from AdC/EBP-treatment or AdGFP-treatment mice were stained by immunohistochemistry for p27and Hesl. 6. Statistical analysisStatistical analyses were performed with SPSS software (16.0version), with a P value<0.05considered significant and P<0.01considered very significant.[Results]1. C/EBPa was downregulated in hepatoma cell lines and human HCC tissues.The expression of C/EBPa was different significantly in human mature hepatocytes and hepatoma cell lines. The level of C/EBPa expression was the highest in human mature hepatocytes, whereas C/EBPa expression was severely repressed in all hepatoma cells. Real-time PCR analysis showed that more than half of HCC patients were accompanied with reduced expression of C/EBPa compared with their surrounding tissues. And immunohistochemistry analysis showed the expression of C/EBPa protein reduced in4HCC tissues compared with the adjacent tissues.2. Construction of recombinant replication-deficient adenovirus.The recombinant plasmids pAdC/EBPa was established by homologous recombination and confirmed by restriction endonuclease digestion and sequencing. GFP expression could be observed at the fourth day after packing of the linearized pAdC/EBPa. In293cells,1×1010efu/ml titer of AdC/EBPa.was obtained after amplification.3. Over-expression of C/EBPa inhibits the tumorigenesis of hepatoma cells in vitro and in vivo.More than95%of human hepatoma Huh7cells expressed GFP after infected by AdC/EBPa for72h. The expressions of C/EBPa mRNA and protein were up-regulated significantly compared with the control cells. Furthermore, immunofluorescence showed that the expression of C/EBPa was located in cell nucleus.Delivery of C/EBPa in Huh7cells evoked the expression of a package of hepatocyte marker genes, including CYP3A7, CYP3A4, CYP2C9, CYP2D6, PEPCK, G-6-P, GYS2, CPS1and UGT1A6, whereas the level of ALB, APROAI and GS had no significant change. In contrast, knockdown of C/EBPa by siRNA decreased the expression of hepatocyte marker genes, including PEPCK, CYP3A4, CPS1, OAT, ALB, GS, TTR.Forced re-expression of exogenous C/EBPa significantly suppressed the proliferation of Huh7cells at the third day after infection, compared with the control infected with AdGFP (P<0.05). In addition, the expression of AdC/EBPa dramatically reduced the number and size of surviving colonies. Cell cycle analysis by flow cytometry revealed that Huh7cells infected by AdC/EBPa were arrested at the Gl/S phase. On the4th day after C/EBPa delivery,17.95±2.55%of AdC/EBPa-infected Huh7cells were in S phase, while35.07±1.26%of AdGFP-infected cells was detected in S phase at the same time points (P<0.01).To further confirm the effect of C/EBPa on HCC cells, we isolated primary cancer cells and TAFs from two human HCC tissues, respectively. In vitro ATP-tumor chemosensitivity assay demonstrated that administration of C/EBPa suppressed the proliferation of cancer cells and TAFs after7days infection in a dose-dependent manner (P<0.05). Additionally, we examined the anti-tumorigenicity effect of C/EBPa in vivo. The tumor nodules were first detected as early as week3postinoculation in mice bearing Huh7xenografts expressing GFP and all the nude mice developed tumor nodules at week5. Meanwhile, no identifiable tumors were observed in mice bearing Huh7cells infected by C/EBPa up to6weeks. These data demonstrate that C/EBPa abolishes the tumorigenicity ability of Huh7cell both in vitro and in vivo.4. C/EBPa inhibits HCC xenograft growth in mice.We used two different xenograft models to investigate whether over-expression of AdC/EBPa could impede the growth of an established tumor xenograft in nude mice.Intratumor injection of AdC/EBPa at day21postinoculation resulted in reduced tumor volume and tumor weight of Huh7cells xenograft in comparison with AdGFP treatment (P<0.05). In addition, an orthotopic HCC model was used to further evaluate the anti-tumor effect of C/EBPa. Interestingly, we found that only60%of the mice (3of5) administrated with AdC/EBPa developed tumor nodules in the liver after3weeks treatment, whereas no tumor in the liver was observed in the other two mice. In contrast, all the mice treated with AdGFP developed gross tumor nodules in the liver which were larger than those of mice receiving AdC/EBPa. In additional, no ascites was detected in mice receiving AdC/EBPa, whereas2of the5mice receiving AdGFP developed severe hemorrhagic ascites. Moreover, the expression of PCNA and Ki67in liver was significantly suppressed in AdC/EBPa group compared with AdGFP group as demonstrated by immunohistochemical staining.5. Over-expression of C/EBPa inhibits Notchl/HES1/p27cascades.To observe the effect of C/EBPa on Notch signaling, we monitored the levels of Notchl signaling. Both immunocytochemical staining and Western blot analysis unraveled that C/EBPa administration resulted in significant decrease of Notchl expression in Huh7cells. Further analysis revealed that Notchl was reduced in nucleus upon C/EBPa Treatment, whereas the cytoplasm Notchl remained unchanged. It is proven that the translocation of the Notch intracellular domain (NICD) into the nucleus will activate transcription of several target genes. Our data showed that C/EBPa treatment suppressed the mRNA expression of Hesl, c-Myc and cyclin D1.Western blot and immunofluorescence staining indicated forced expression of C/EBPa resulted in reduction of Hesl along with induction of p27, suggesting that C/EBPa could inhibit the activation of Notchl signaling.To further explore the role of Notchl signaling in the anti-tumor effect of C/EBPa, we transfected pcDNA3.0-Hesl into Huh7cells. The adenovirus was then added6h later. CCK-8analysis showed that anti-proliferation effect of C/EBPa was significantly blocked by up-regulation of Hes1. Moreover, the reduction of Hesl by C/EBPa was abrogated with the expression of Notchl. To confirm the effect of C/EBPa on Notchl signaling, the expression of Hesl and p27was assessed in an orthotopic tumor xenograft model by immunohistochemistry. Hesl expression was significantly reduced, while p27expression was induced in liver xenograft tumors from mice treated with AdC/EBPa in compared with those from mice treated with AdGFP.[Conclusions]1. The expression of C/EBPa was remarkly down-regulated in the hepatoma cell lines and most human HCC tissues.2. The recombinant replication-deficient adenovirus AdC/EBPa constructed by AdEasyTM system could express C/EBPa efficiently.3. Force re-expression of C/EBPa up-regulates the expression of some liver-specific genes, which results in the Gl/S arrest, inhibits cell proliferation and reduces the colony formation ability in hepatoma cell lines, abolishes the tumorigenicity of hepatoma cells.4. Overexpression of C/EBPa successfully suppresses the growth of experimental liver tumor in vivo.5. Overexpression of C/EBPa regulate tumor growth through inhibits Notch1/HES1/p27cascades.6. C/EBPa might serve as a novel effective agent for the treatment of HCC in future.