Mechanism Study Of Hedgehog Signaling Regulating Twist1 Expression And Promoting Hepatic Cellular Cancer Invasion And Metastasis

Background:Primary hepatocellular carcinoma(HCC) is one of common human malignancies. It is the third most deadly and the fifth most common malignancy worldwide, with an estimation of 600000 new cases per year. Surgical removal and liver transplantation remain are the most effective therapy for HCC. Although various adjuvant approaches to treat non-resectable HCC are available, the efficacy and three-year survival rate(30–40%) are not promising. Invasion and metastases are two fundamental properties of the malignancy, which determine the prognosis of HCC patients, especially poorly differentiated HCC. Thus, seeking molecular targets that could improve the treatment outcome is intriguing.The invasive mechanism of hepatocellular carcinoma is still not clear. The existing theory of tumorigenesis, such as genetic changes, including the activation of proto-oncogenes, gene deletion or mutation, the reactivation of telomerase activity and epigenetic abnormalities, can not be the comprehensive explanation of molecular mechanisms to metastasis and recurrence of hepatocellular carcinoma.The present study showed that tumor cells have obvious heterogeneity. In the same tumor cells, many cells showed obvious inconsistency in genotype and molecular phenotype, allowing the the differences in growth, invasion and metastasis ability of these tumor cells, and their sensitivity to drugs.The hedgehog(Hh) signaling pathway is highly conserved, and has a crucial role in embryogenesis, adult tissue homeostasis and carcinogenesis. Transcription factors Gli-1, Gli-2 and Gli-3 constitute the Gli family. Gli-1/2 have been implicated to be pivotal for positively transactivating target genes, such as snail, Gli-2, c-myc and B-cell lymphoma-2, and cyclin D and so on. Hh signaling is activated following binding of Sonic, Desert or Indian Hh ligands to their transmembrane receptor Patched, leading to the release of Gli transcription factors by derepressing Smoothened(Smo). Gli-1/2 translocate to the nucleus, where they bind to the Gli-binding consensus element in target genes resulting in their activation. A novel tGli1 isoform in which 41 amino acids are deleted, corresponding to an alternative splicing of the entire exon 3 and part of exon 4 of the gene was reported in glioblastoma and breast cancer.Hh signaling regulates the expression of Gli1 isoforms. Most notably, the tGli1 isoform behaves as a gain-offunction of Gli1 and induces expression of genes that are normally not regulated by Gli1, and it is known to foster more aggressive cancer phenotypes. tGli1 can promote glioblastoma and breast cancer cell migration and invasion by enhancing the expression of MMP-2 and MMP-9. Overexpression and aberrant activation of Hh signaling molecules have been demonstrated in 50–70% of hepatoblastomas, primary HCC and cholangiocyte carcinomas, and the Smo antagonists, cyclopamine and GDC-0449 have been used to block the growth of hepatoma cell-derived xenografts in animal experiments. However, no study is available focusing on whether overexpression of Hh molecules or enhancing Hh signaling activity is responsible for the highly metastatic behavior of HCC.Epithelial–mesenchymal transition(EMT) is characterized as the loss of epithelial morphology and development of a highly mobile mesenchymal phenotype. During cancer progression, EMT promotes the metastatic capability of cancer cells. Characteristic changes in the EMT process include: losing the expression of E- Cadherin which is the marks of epithelial cells, the nuclear translocation of β-catenin, and raising the expression of N-Cadherin, vimetin, etc, which are the markers of mesenchymal cells. E-Cadherin is an important adhesion molecule of epithelial cells. It is one of the important moleculars that can maintain epithelial phenotype, by bonding with extracellular immune globulin structure domain to form linkage, and linking to actin cytoskeleton through the α、β-catenin in the cytoplasm to form cell tight junction between cells. The decreased expression of E-Cadherin in tumor cells can reduce the cell adhesive ability, and promote tumor cells detached from the original site to invade surrounding tissues.Objective:This project intends to research the role of Hh signaling pathway in the regulation of invasion and metastasis of HCC, to explore the molecular mechanism and targets. We want to evaluate the function of key molecular Gli1 in clinical treatment and prognosis judgement in HCC. The results help to further study in the nature of metastasis of hepatoma, and to provide a new theoretical basis to target Hh signaling in HCC therapy.Methods:1. Flow cytometry was applied to analyze and enrich the subpopulation of hepatoma cells according to their surface markers CD133 and EpCAM: CD133+/EpCAM+ or CD133-/EpCAM- subpopulations.2. Clarify of the phenotypes of the different subpopulation of hepatoma cells including tumor formation, the sensitivity of chemotherapy, and metastasis ability.3. In vivo experiments were applied to confirm highly invasive capability of CD133-/EpCAM- subpopulation hepatoma cells by establishing an orthotopic transplantation model of hepatocellular cells in immunodeficiency NOD-SCID mice using in vivo bioluminescent imaging system, and to confirm the invasion and metastasis of transplanted tumor after using Hh signaling pathways inhibitor.4. Luciferase reporter assays experiment conformed activation of Hh signaling pathways in the different the subpopulation of hepatoma cells. The application of Hh signaling pathway inhibitor confirmed that the aberrant hedgehog signaling can promote the invasion and metastasis of hepatocellular carcinoma cells.5. To obtain ahighly invasive capability subpopulation hepatoma cells by using transwell assay. And flow cytometry was applied to test the markers of these cells.6. Double immunofluorescence revealed the cellular localization of E-Cadherin and Vimetin in the different subpopulation of hepatoma cells. Real-time PCR and Western blot were applied to validate the content of Gli in mRNA and protein levels.7. Real-time PCR and Western blot were applied to validate the content of Twist and Snail in mRNA and protein levels in different subpopulation cells..8. Real-time PCR was applied to validate the content change of Gli1、Twist、Snail and MMPs after using Hh signaling pathways inhibitor in different subpopulation cells..9. Dual luciferase reporter assays were used to test if Gli1 can promote Twist promoter luciferase activity in different subpopulation cells.10. Bioinformatics prediction techniques were used to predict the potential binding sites of Gli1 and Twist and biology relationship, looking for the molecular mechanism of adjusting Twist1 expression byGli1.11. Regular RT–PCR assay was applied to investigate tGli1 expression in several hepatoma cell lines and primary human hepatocytes.Results:1. We first separated CD133+/EpCAM+ and CD133-/EpCAM- subpopulation of hepatoma cells from Hep3 B, HepG2,Huh-7,HLE,HLF,SKHep1 cell lines. As a result,the CD133+/EpCAM+ subpopulation in Hep3 B,HepG2, and Huh-7 cells were10.3%,2.3%,18.3%. Respectively, the CD133-/EpCAM- subpopulation were 15.09%,72.47%,28.81%. Whereas poorly differentiated HLE, HLF, and SKHep1 cells were almost all negative for these two markers. Culture the sorting cells.2. We obtained a subpopulation CD133-/EpCAM- Trans selected cells which displaying a highly invasive capability by using a transwell approach to select cells(transwell-selected, TS).3. We confimed the different subpopulations of hepatoma cells from the same hepatoma cell line have different phenotype. Tumorigenicity of FACS-enriched subpopulations: CD133+/EpCAM+ subpopulations formed a higher number of spheroids than their double negative counterparts(p <0.001).4. Enhanced chemoresistance in HLE CD133-/EpCAM- subpopulation: more CD133-/EpCAM- subpopulation HLE cells survived in a 24 h exposure to cisplatin, doxorubicin, or sorafenib in comparison to the CD133+/EpCAM+ subpopulations. As the major subpopulation of poorly-differentiated hepatoma cell lines, the chemoresistance of CD133-/EpCAM- subpopulation cells may be comparable in insensitivity to chemotherapy of poorly-differentiated HCC.5. Cell migration capability of Huh-7 subpopulations was evaluated using a transwell Matrigel invasion assay. A marked difference in invasive capacity was found between different subpopulations with the highest rate of migration in the TS CD133-/EpCAM- Huh-7 subpopulation. Enhanced invasive capability decreased in the LDE225- treated subpopulation cells.6. Lentiviral vector LUC–PGK – EGFP was tranfected successfully into Huh-7 Trans selected subpopulation and Huh-7 untreated cells. GFP positive hepatoma cells were enriched by Flow cytometry and transplanted into immunodeficiency NOD-SCID mice in the liver, and an orthotopic transplantation model was established.7. The transplanted tumors were detected by bioluminescent imaging system on the third month after transplantation. Consequently, Huh-7 trans selected subpopulation cells significantly increased the distant metastasis in vivo model compared with the Huh-7 unsorted cell. Hh signailing pathway inhibitor LDE225 rescued the distant metastasis in vivo.8. We transfected the Huh-7 subpopulations with a GLI-lux reporter system, and determined luciferase activity one day after transfection. It was found that luciferase activity was much higher in the Huh-7 CD133-/EpCAM- subpopulation cells than their double-positive counterparts, while cells transfected with mutated GLI-Lux did not show any luciferase activity. Moreover, when transfected cells were treated with a Hh signaling inhibitor, cyclopamine(CPM), both cell proliferation, as determined by SWT-1 reagent, and luciferase activity were markedly inhibited by the treatment.9. We performed immunocyto chemical staining for EMT markers including E-cadherin and vimentin in Huh-7 subpopulations. Strong E-cadherin staining was observed in the Huh-7 CD133+/EpCAM+ subpopulation, whereas, vimentin staining was negative in the cytoplasm of these cells. Meanwhile, E-cadherin was negative in the Huh-7 CD133-/ EpCAM-subpopulation, whereas, vimentin staining was positive. That means the negative subpopulation cells were in the status of EMT. In contrast to DP cells, TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry.10. qPCR evaluated the gene expression of Gli1, Gli2 and Twist in various subpopulations, and found that the mRNA levels of Gli1,Gli2 and Twist were extraordinarily increased in the Huh-7 Trans selected subpopulation compared with the CD133+/EpCAM+ subpopulation. Western blotting analysis results were consistent with those of real-time RT–PCR, and it was evident that these two transcriptional factors, Gli-1/2, were increased in the nuclear fraction of Huh-7 CD133-/EpCAM- subpopulation and Huh-7 Trans selected subpopulation compared with the unsorted or CD133+/EpCAM+ subpopulation. LDE225-treated cells had lower levels of Gli-1/2 or Twist gene expression than those without the treatment.11. To determine the molecular basis for the highly metastatic property of this TS population, we evaluated the gene expression of MMP-1, 2 and 9 in various subpopulations, and found that the mRNA levels of MMP-1, MMP-2 and MMP-9 were extraordinarily increased in the TS Huh-7 subpopulation(4294-, 74- and 36-fold) compared with the double-positive(DP) subpopulation, although a tendency of increased MMP gene expression existed from the DP, unsorted to DN subpopulations.12. Dual luciferase reporter assays confirmed that Gli induced Twsit1 expression at transcriptional level.13. Bioinformatics analysis predicted Twist1 transcription factor binding sites. There are 262 binding sites in the promoter of Twist1. But Gli1 was not found among them. The results revealed Gli1 may not bind with Twist1 promoter directly. Bioinformatics analysis find out that several protein including HOXA5 have binding site in the Twist1 promoter region.14. To investigate tGli1 expression, we examined the tGli1 variant fragment in several hepatoma cell lines and primary human hepatocytes by a regular RT–PCR assay, and found that t Gli1 transcripts were negative in normal liver cells and well differentiated hepatoma cells with a high level of hepatic specific gene expression, including Hep3 B and HepG2 cells. However, tGli1 was positive in the poorly differentiated HLF line and high metastatic Huh-7 subpopulations, including the TS Huh-7 cells.Conclusions:1. Hepatoma exhibits a series of heterogeneous subpopulations in its cell surface markers, tumorigenicity, invasion and metastatic capability. The heterogeneity of hepatoma cells determined the different phenotypes of each subpopulation from the same hepatoma cell line. CD133 and EpCAM were useful markers to identify hepatoma cells. CD133+/EpCAM+ subpopulation cells appeard to form a higher number of spheroids, and reduce the chemoresistance. Whereas, enhanced invasive capability was detected in CD133-/EpCAMsubpopulation cells.2. Hedgehog signailing pathway promoted CD133-/EpCAM- subpopulation cells invasion and metastasis in vitro and invo. The Hh signailing inhibitor LDE225 could rescue the invasion and metastasis of CD133-/EpCAM- subpopulation cells in vitro and in vivo.3. The highly metastatic capability of D133-/EpCAM- subpopulations was highly attributed to significant epithelial–mesenchymal transition, enhanced Hh activity and aberrant occurrence of a tGli1 variant, which appears to be responsible for the highly invasive behavior.