Murine HCC Cell Research Articles

Lactobacillus acidophilus suppresses non-alcoholic fatty liver disease-associated hepatocellular carcinoma through producing valeric acid

Gut probiotic depletion is associated with non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). Here, we investigated the prophylactic potential of Lactobacillus acidophilus against NAFLD-HCC. NAFLD-HCC conventional and germ-free mice were established by diethylnitrosamine (DEN) injection with feeding of high-fat high-cholesterol (HFHC) or choline-deficient high-fat (CDHF) diet. Orthotopic NAFLD-HCC allografts were established by intrahepatic injection of murine HCC cells with HFHC feeding. Metabolomic profiling was performed using liquid chromatography-mass spectrometry. Biological functions of L.acidophilus conditional medium (L.a CM) and metabolites were determined in NAFLD-HCC human cells and mouse organoids. L.acidophilus supplementation suppressed NAFLD-HCC formation in HFHC-fed DEN-treated mice. This was confirmed in orthotopic allografts and germ-free tumourigenesis mice. L.a CM inhibited the growth of NAFLD-HCC human cells and mouse organoids. The protective function of L.acidophilus was attributed to its non-protein small molecules. By metabolomic profiling, valeric acid was the top enriched metabolite in L.a CM and its upregulation was verified in liver and portal vein of L.acidophilus-treated mice. The protective function of valeric acid was demonstrated in NAFLD-HCC human cells and mouse organoids. Valeric acid significantly suppressed NAFLD-HCC formation in HFHC-fed DEN-treated mice, accompanied by improved intestinal barrier integrity. This was confirmed in another NAFLD-HCC mouse model induced by CDHF diet and DEN. Mechanistically, valeric acid bound to hepatocytic surface receptor GPR41/43 to inhibit Rho-GTPase pathway, thereby ablating NAFLD-HCC. L.acidophilus exhibits anti-tumourigenic effect in mice by secreting valeric acid. Probiotic supplementation is a potential prophylactic of NAFLD-HCC. Shown in Acknowledgments.

Radiosensitization effect by HDAC inhibition improves NKG2D-dependent natural killer cytotoxicity in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Radiotherapy (RT) controls HCC unsatisfactorily and temporarily. Histone deacetylase inhibitor (HDACi) is a heterogeneous group of epigenetic therapeutics with promising anticancer effects and synergism in combination with RT. HDACi modulates natural killer (NK) cell ligand expression on tumor cells, and leads to immune evasion of cancer cells. Expressions of NK group 2D (NKG2D) ligands on cancer cells determine the cytotoxic effect by interacting with NKG2D receptor on NK cells. However, the role of NKG2D signaling in HCC upon combined RT and HDACi remains unclear.Method In vitro co-culture system with NK cells was tested for human and murine HCC cell lines. Pan-HDACi (panobinostat) and specific HDAC4 knockdown (HDAC4-KD) were used for HDAC inhibition. Clonogenic assay and flow cytometry examined HCC cell survival and NKG2D ligand expression, respectively. Syngeneic mouse model was used to validate the radiosensitizing effect in vivo.ResultsCombined RT and HDACi/HDAC4-KD significantly enhanced NK cell-related cytotoxicity and increased NKG2D ligands, MICA/MICB expressions in human and RAE-1/H60 expressions in murine HCC cells. Delayed tumor growth in vivo by the combinational treatment of RT and HDACi/HDAC4-KD was shown with the associated NKG2D ligand expressions. However, NKG2D receptor did not significantly change among tumors.ConclusionRadiosensitizing effect with combined RT and HDAC inhibition increased the expression of NKG2D ligands in HCC cells and enhanced their susceptibility to NK cell-mediated cytotoxicity. These findings imply the potential use of combined RT/HDACi and NK cell-directed immunotherapy.

NOX1 inhibition attenuates the development of a pro\u2010tumorigenic environment in experimental hepatocellular carcinoma

BackgroundThe poor prognosis of advanced HCC and limited efficacy of current systemic treatments emphasize the need for new or combined targeted therapies. The development of HCC is a multistage process in which liver injury appears in a complex microenvironment associated with oxidative stress. NOX enzymes are the main source of ROS during hepatocarcinogenesis and NOX1 in particular has shown correlation with poor prognosis of HCC patients. This study evaluates the effect of pharmacological NOX1 inhibition on the development and progression of HCC and its effect on the tumor microenvironment.MethodsThe in vitro cytotoxic effects of the NOX1 inhibitor GKT771 (Genkyotex) on human Huh7 and Hep3B and murine Hepa1-6 HCC cell lines, the human THP1 monocyte cell line and mouse macrophages were evaluated via MTT, LDH activity and CaspGlo® assays. In order to induce in vivo HCC, male SV129 wild-type mice received weekly IP injections of diethylnitrosamine (DEN) (35 mg/kg) for 20–25 weeks. Mice were treated with vehicle or GKT771 (30 mg/kg) via oral gavage, daily or twice daily, in preventive and therapeutic studies. The liver damage was evaluated for inflammation, angiogenesis, fibrosis and HCC development via histology, RT-qPCR, multiplex analyses and ROS levels.ResultsA concentration-dependent reduction in cellular activity of the human HCC cell lines without cytotoxicity was observed. GKT771 treatment reduced LPS-induced pro-inflammatory bone-marrow derived macrophage polarization. DEN injections resulted in 100 % tumor formation and the induction of HCC markers which could be reduced by twice daily dosing of GKT771 at early onset of advanced HCC. DEN-induced HCC resulted in an upregulation of pro-inflammatory, angiogenic and fibrotic markers which was less pronounced in GKT771 treated mice in all treatment regimens. In line, liver fibrosis was induced in HCC mice and this to a lesser extend upon GKT771 treatment.ConclusionsNOX1 inhibition showed to be safe and well tolerated and was able to attenuate the induction of a pro-inflammatory, angiogenic and pro-fibrotic microenvironment suggesting that this might be a promising adjuvant therapeutic strategy in the treatment of advanced HCC.

SILAC proteomics implicates SOCS1 in modulating cellular macromolecular complexes and the ubiquitin conjugating enzyme UBE2D involved in MET receptor tyrosine kinase downregulation

Suppressor of Cytokine Signaling 1 (SOCS1) functions as a tumor suppressor in hepatocellular carcinoma and many other types of cancers. SOCS1 mediates its functions by inhibiting tyrosine kinases, promoting ubiquitination and proteasomal degradation of signal transducing proteins, and by modulating transcription factors. Here, we studied the impact of SOCS1 on the hepatocyte proteome using Stable Isotopic Labelling of Amino acids in Cell culture (SILAC)-based mass spectrometry on the Hepa1-6 murine HCC cell line stably expressing wildtype SOCS1 or a mutant SOCS1 with impaired SH2 domain. As SOCS1 regulates the hepatocyte growth factor (HGF) receptor, the MET receptor tyrosine kinase (RTK), the SILAC-labelled cells were stimulated or not with HGF. Following mass spectrometry analysis, differentially modulated proteins were identified, quantified and analyzed for pathway enrichment. Of the 3440 proteins identified in Hepa-SOCS1 cells at steady state, 181 proteins were significantly modulated compared to control cells. The SH2 domain mutation and HGF increased the number of differentially modulated proteins. Protein interaction network analysis revealed enrichment of SOCS1-modulated proteins within multiprotein complexes such as ubiquitin conjugating enzymes, proteasome, mRNA spliceosome, mRNA exosome and mitochondrial ribosome. Notably, the expression of UBE2D ubiquitin conjugating enzyme, which is implicated in the control of growth factor receptor tyrosine kinase signaling, was found to be regulated by SOCS1. These findings suggest that SOCS1, induced by cytokines, growth factors and diverse other stimuli, has the potential to dynamically modulate of large macromolecular regulatory complexes to help maintain cellular homeostasis.

CKD-5, a novel pan-histone deacetylase inhibitor, synergistically enhances the efficacy of sorafenib for hepatocellular carcinoma

BackgroundHistone deacetylase inhibitors (HDACIs) have distinctive epigenetic targets involved in hepatocarcinogenesis and chemoresistance. A recent phase I/II study reported the possibility of HDACI as a chemosensitizer in sorafenib-resistant patients. In this study, we evaluated whether CKD-5, a novel pan-HDACI, can potentiate the efficacy of sorafenib.MethodsThe anticancer effect of CKD-5 with and without sorafenib was evaluated in vitro using an MTS assay with human HCC cells (SNU-3058 and SNU-761) under both normoxic and hypoxic conditions. Microarray analysis was performed to investigate the mechanism of cell death, which was also evaluated by small interfering RNA (siRNA) transfection and subsequent immunoblot assays. In vivo experiments were conducted using two different murine HCC models. C3H mice implanted with MH134 cells and C57BL/6 mice implanted with RIL-175 cells were treated with weekly CKD-5 with and without sorafenib for 2 weeks.ResultsCKD-5 treatment significantly suppressed human HCC cell growth in both normoxic and hypoxic conditions. Microarray analysis and real-time PCR showed that CKD-5 treatment significantly increased peripherin expression in HCC cells and that downregulation of peripherin by siRNA decreased CKD-5-induced apoptosis. The combination of CKD-5 and sorafenib decreased cell viability more effectively than sorafenib or CKD-5 monotherapy in human and murine HCC cells. The effectiveness of the combination therapy was consistently demonstrated in the animal models. Histological and biochemical analyses demonstrated good tolerance of CKD-5 plus sorafenib in vivo.ConclusionCKD-5 may enhance sorafenib efficacy through epigenetic regulation. The combination of CKD-5 and sorafenib might be a novel therapeutic option for the treatment of HCC.

Hypoxia-induced HMGB1 expression of HCC promotes tumor invasiveness and metastasis via regulating macrophage-derived IL-6

Hypoxia is associated with the progression of hepatocellular carcinoma through promotion of spontaneous metastasis but the mechanism remains unclear. Here, we hypothesis that tumor cell-derived HMGB1 orchestrates macrophages infiltration and promotes metastasis of HCC via enhancing macrophage-secreted IL-6 under hypoxia. HMGB1 expression was robustly exacerbated in tumors of HCC patients with PVTT. Meanwhile, hypoxia exposure gave rise to HMGB1 expression in hepatoma cells of human and mouse in a HIF-1α-dependent manner and subsequently induced the infiltration and reprogramming of macrophages to augment the expression of Il-6. Further study demonstrated macrophage-derived IL-6 enhanced the invasiveness and metastasis of murine HCC cells. Therefore, our study provides a novel understanding of the relationship between tumor cells and tumor associated macrophages (TAMs) in the context of hypoxia.

Sulfatase-1 knockdown promotes in vitro and in vivo aggressive behavior of murine hepatocarcinoma Hca-P cells through up-regulation of mesothelin.

Our previous study (Oncotarget 2016; 7:46) demonstrated that the over-expression of sulfatase-1 in murine hepatocarcinoma Hca-F cell line (a murine HCC cell with lymph node metastatic [LNM] rate of >75%) downregulates mesothelin and leads to reduction in lymphatic metastasis, both in vitro and in vivo. In current work, we investigated the effects of Sulf-1 knockdown on mesothelin (Msln) and it's effects on the in vitro cell proliferation, migration, invasion, and in vivo tumor growth and LNM rate for Hca-P cells (a murine HCC cell with LNM rate of <25%). Western blotting and qRT-PCR assay indicated that both in vitro and in vivo Sulf-1 was down-regulated by 75% and 68% and led to up regulation of Msln by 55% in shRNA-transfected-Sulf-1-Hca-P cells compared with Hca-P and nonspecific sequence control plasmid transfected Hca-P cell (shRNA-Nc-Hca-P). The in vitro proliferation, migration and invasion potentials were significantly enhanced following Sulf-1 stable down-regulation. In addition, Sulf-1 knock-down significantly promoted tumor growth and increased LNM rates of shRNA-Sulf-1-Hca-P-transplanted mice by 78.6% (11 out of 14 lymph nodes were positive of cancer). Consistent with our previous work, we confirmed that Sulf-1 plays an important role in hepatocarcinoma cell proliferation, migration, invasion and metastasis. The interaction between Sulf-1 and Msln is a potential therapeutic target in the development of liver cancer therapy.

Cross-talk between TNF-α and IFN-γ signaling in induction of B7-H1 expression in hepatocellular carcinoma cells.

Clinical benefit from immunotherapy of B7-H1/PD-1 checkpoint blockade indicates that it is important to understand the regulatory mechanism of B7-H1 expression in cancer cells. As an adaptive response to the endogenous antitumor immunity, B7-H1 expression is up-regulated in HCC cells. B7-H1 expression is induced mainly by IFN-γ released from tumor-infiltrating T cells in HCC. In addition, HCC is a prototype of inflammation-related cancer and TNF-α is a critical component of inflammatory microenvironment of HCC. In the present study, we asked whether TNF-α can promote the expression of B7-H1 induced by IFN-γ in HCC cells. We found that JAK/STAT1/IRF1 was the primary pathway responsible for induction of B7-H1 expression by IFN-γ in human HCC cell lines. TNF-α and IFN-γ synergistically induced the expression of B7-H1 in the HCC cells. Moreover, the mechanism of the synergy was that TNF-α enhanced IFN-γ signaling by upregulating the expression of IFN-γ receptors. Furthermore, B7-H1 expression induced synergistically by TNF-α and IFN-γ in murine HCC cells facilitated tumor growth in vivo. Our findings suggest that TNF-α may enhance the adaptive immune resistance mediated by IFN-γ-induced B7-H1 in HCC cells.

Abstract LB-005: TRIP13/PCH2 is a novel regulator of EGF-mTOR signaling promoting hepatocellular carcinoma development

Abstract Hepatocellular carcinoma (HCC) is currently the third most common cause of cancer-related mortality with limited treatment options and bleak prognosis. The main risk factors for HCC have been linked to hepatitis infection, alcoholic liver injury, non-alcoholic fatty liver disease (NAFLD) and metabolic diseases. Current therapeutic options are limited, which could be due to the lack of understanding of the key cellular players and signalling pathways in HCC. In order to find novel regulators of HCC development, we surveyed expression data of human hepatocellular carcinoma (E-GEOD-25097) and found the AAA-ATPase Thyroid Hormone Receptor Interactor 13 (TRIP13) to be upregulated in tumorous tissue compared to non-tumorous tissue. Consistently, using a mouse model for HCC by injection of Diethylnitrosamine (DEN), we observed induced expression of TRIP13 in liver tumors compared to healthy liver tissue. Additionally, in a model of liver regeneration by partial hepatectomy, induction of TRIP13 coincided with a peak in cell proliferation in the regenerating liver. Strinkingly, Adeno-associated virus (AAV)-mediated hepatocyte-specific knockdown of TRIP13 impeded liver regeneration upon partial hepatectomy, suggesting an important role of TRIP13 in liver cell proliferation. Indeed, knockdown (KD) of TRIP13 severely impaired cell growth and proliferation in different human and murine HCC cell lines in vitro. To investigate the oncogenic potential of TRIP13 in vivo, we injected lentiviral infected cells subcutaneously into mice and measured tumor growth. Remarkably, knockdown of TRIP13 in Hepa1-6 cells led to the development of smaller tumors compared to control cells, whereas TRIP13-deficient Huh7 cells did not form any tumors. Interestingly, shRNA knockdown of TRIP13 in HCC cell lines showed decreased phosphorylation of key players in the mTOR signalling pathway and reduction of the downstream Cyclin D1 levels. Concurrently, the mRNA and protein expression of EGF receptor was also downregulated in TRIP13 KD-cells. Taken together, we identified a novel role of TRIP13 during hepatocyte proliferation both in liver regeneration and HCC. Additionally, our findings also support an oncogenic function of TRIP13 through the regulation of the EGFR-mTOR signalling axis. Citation Format: Bettina Meissburger, Jessica Chan, Roldan de Guia, Stephan Herzig, Mauricio Berriel Diaz. TRIP13/PCH2 is a novel regulator of EGF-mTOR signaling promoting hepatocellular carcinoma development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-005.

Targeting of tumour-infiltrating macrophages via CCL2/CCR2 signalling as a therapeutic strategy against hepatocellular carcinoma

ObjectiveHepatocellular carcinoma (HCC) is an aggressive malignancy with limited effective treatment options. An alternative strategy is to target cells, such as tumour-infiltrating macrophages, in the HCC tumour microenvironment. The CCL2/CCR2...

CRKL knockdown promotes in vitro proliferation, migration and invasion, in vivo tumor malignancy and lymph node metastasis of murine hepatocarcinoma Hca-P cells

Our previous study (Biomed Pharmacother 2015;69:11) demonstrated that the over-expression of CRKL, a chicken tumor virus number 10 regulator of kinase-like protein, suppresses in vitro proliferation, invasion and migration of murine hepatocarcinoma Hca-P cell, a murine HCC cell with lymph node metastatic (LNM) rate of ∼25%. In current work, we investigated the effects of CRKL knockdown on the in vitro cell proliferation, migration and invasion, and on the in vivo tumor malignancy and LNM rate and level for Hca-P cells. Western blotting assay indicated that CRKL was down-regulated by ∼90% in a monoclonal CrkL-shRNA-transfected Hca-P cells. Compared with Hca-P and unrelated-shRNA-transfected Hca-P cell, the in vitro proliferation, migration and invasion potentials were significantly enhanced following CRKL stable deregulation. CRKL knock-down significantly promoted the tumorigenicity malignancy, LNM rates and level of Hca-P-transplanted mice. Consistent with our previous work, it can be concluded CRKL plays an important role in hepatocarcinoma cell proliferation, invasion and migration as well hepatocarcinoma malignancy and metastasis. It functions as a potential tumor suppressor in hepatocarcinoma.

CRKL overexpression suppresses in vitro proliferation, invasion and migration of murine hepatocarcinoma Hca-P cells

The signal adaptor CRK family protein play important roles in cancer cell progression, proliferation, migration and invasion. Previously, we showed that CRK was involved in lymphatic metastatic potential of murine hepatocarcinoma cells. In current work, as a member of CRK family, chicken tumour virus number 10 regulator of kinase-like protein (CRKL) was revealed to be associated with malignant behaviors of Hca-P, a murine HCC cell with lymph node metastatic (LNM) rate of ∼25%. CRKL overexpression in Hca-P by a constructed eukaryotic expression vector of pcDNA3.1/V5-HisB-CRKL significantly ameliorated its malignant biological properties. CCK-8 and soft agar colony formation assays indicated CRKL overexpression significantly inhibits the cell proliferation and colony formation abilities of Hca-P. Additionally, transwell assays indicated that the Hca-P cell migration and invasion capacities were apparently reduced following CRKL overexpression. As Hca-P is an ideal hepatocarcinoma cell model with low (initial) LNM potential, CRKL is shown to act as a potential suppressor and to provide new insight for both the malignant behaviors of hepatocarcinoma cells and lymphatic metastasis mechanism of hepatocarcinoma.

Abstract 721: Suppression of orthotopic liver cancers by a combinatorial therapy of radiation and adenoviral vectors expressing interleukin 12

Abstract Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. Because of difficulty in early diagnosis, only a minority of HCC patients are candidates for resection or transplantation, the traditional curative treatments. Recent development of image-guided radiotherapy and stereotactic radiotherapy offer an alternative therapeutic option for HCC patients with advanced diseases. In addition to eradicate tumor cells via DNA double-strand breaks or induction of apoptosis, radiotherapy also induces immunogenic tumor cell death, releasing a variety of danger-signaling molecules, which may help induce adaptive immune responses against the irradiated local tumors as well as the non-irradiated metastatic tumors. To further increase the therapeutic effect and enhance the antitumor immune responses, we proposed to combine radiation therapy with interleukin 12 (IL-12), a cytokine which has the ability to sensitize suppressive tumor-associated bone marrow-derived stromal cells, enhancing their ability to stimulate CD4+ and CD8+ T cell responses. BALB/c mice were injected at the left liver lobe of 3 x 105 BNL cells, a murine HCC cell line. At day 10, when the tumor volume reaching about 50 mm3, the hepatic tumors were receiving the following treatment: (1) a single dose of radiation of 10 Gy delivering at a mean dose rate of 6.72 Gy/min, (2) a single intratumoral injection of 108 p.f.u. of adenoviral vectors encoding a single-chain murine IL-12 (Ad/IL-12), (3) a combination therapy of 10 Gy radiation and 108 p.f.u. Ad/IL-12, and (4) no treatment. At day 30, mice were sacrificed and the tumor volume was measured by a caliper. The tumors in the control none treated group grew progressively with a mean tumor volume of 2,797 ± 304 mm3. The single treatment of radiation or Ad/IL-12 resulted in objective tumor growth suppression, reducing the mean tumor volume to 367 ± 203 mm3 and 241 ± 204 mm3, respectively, but none of the tumors were regressed after the single agent treatment. In contrast, the combinatorial treatment of radiation and Ad/IL-12 led to a partial (&amp;gt; 50% regression) or complete tumor regression in 80% (8 of 10) of tumors and substantially delayed tumor growth in the tumor-bearing mice. Our preliminary data also showed that the combinatorial treatment of radiation and Ad/IL-12 induced antitumor immune responses and suppressed systemic metastasis. Mechanistic studies of the combinatorial treatment of radiation and Ad/IL-12 are under investigation. Our results suggest that a combinatorial treatment of radiation and IL-12 may represent an alternative therapy for advanced HCC. Citation Format: Mi-Hua Tao, Wen-Shan Tsao, Ping-Yi Wu. Suppression of orthotopic liver cancers by a combinatorial therapy of radiation and adenoviral vectors expressing interleukin 12. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 721. doi:10.1158/1538-7445.AM2014-721

TRC105 for the treatment of hepatocellular carcinoma: Preclinical data and preliminary results from two clinical trials evaluating monotherapy and combination with sorafenib.

211 Background: Endoglin (CD105), is an endothelial cell membrane receptor highly expressed on proliferating tumor vasculature, including in HCC. CD105 is essential for angiogenesis and its expression is upregulated by hypoxia and VEGF inhibition. TRC105 is a chimeric IgG1 anti-CD105 monoclonal antibody that acts by endothelial cell growth inhibition, ADCC and apoptosis. Methods: Preclinical: Murine BNL HCC cells were grown subcutaneously in wildtype Balb/c mice and treated with antibody to murine CD105, (clone MJ7/18; 10-200ug) ± sorafenib (10 mg/kg daily p.o.) or sorafenib alone. Clinical: Patients with HCC (Childs Pugh A/B7), ECOG 0/1, were enrolled in a phase I study of TRC105 at 3, 6, 10, 15mg/kg q 2wks plus sorafenib 400mg bid; or (if sorafenib-refractory) a phase II study of TRC105 at 10 or 15mg/kg q 2wks. Correlative biomarkers: DCE-MRI; FDG-PET; color Doppler ultrasonography; circulating endothelial and endothelial progenitor cells, plasma levels of angiogenic factors; soluble CD105 and tumor IHC for CD105. Results: Preclinical studies: CD105 expression was increased in BNL HCC tumors of mice treated with sorafenib. Anti-CD105 + sorafenib was more effective than sorafenib alone. Clinical trials (N=16): M:F 13:3; Median age = 55 (range 24-67); Phase I with sorafenib (N=8): 1 DLT (increased AST) occurred at 10mg/kg. The most frequent toxicity has been epistaxis. One patient with coronary stenosis developed a fatal MI and one patient with thrombocytopenia developed G3 cerebral tumor hemorrhage. Phase II (N=8): No grade 3/4 treatment-related toxicities . Most frequent toxicities headache (G2; N=3) and epistaxis (G1; N=4). No patient was progression free at 4 months. Median time on study: Phase I- 4 months; Phase II - 2 months. Preliminary evidence of biologic response seen on DCE-MRI in 1pt (of 3 evaluable) with a reduction in Ktrans and kep and 3pts (of 3 evaluable) with reduction in intra-tumoral color flow on Doppler. Conclusions: TRC105 is well tolerated both as single agent and combined with sorafenib. Evidence of biological activity was observed. Full preclinical, clinical and correlative data will be presented. Clinical trial information: NCT01306058, NCT01375569.

Abstract 2413: Human glypican-3 promotes hepatocellular carcinoma progression via induction of epithelial-mesenchymal transition

Abstract Hepatocellular carcinoma (HCC) is the third most important cause of cancer mortality worldwide. Epithelial-mesenchymal transition (EMT) has been shown to be an important biological process involved in the development and progression of HCC. The factors driving this process, however, are still largely unclear. Glypican-3 (GPC3) is upregulated in HCC and this is associated with poor prognosis. The details of the biological role of GPC3 in HCC are, however, yet to be fully elucidated. We hypothesized that GPC3 may promote HCC progression by inducing EMT. In this study we have examined the effect of human GPC3 in the progression of HCC using a murine HCC cell line (1MEA) derived from Balb/c mouse. We observed that the 1MEA cell line does not express GPC3 and we stably transfected human GPC3 into this cell line. 1MEA cells stably transfected with human glypican-3 (1MEAhGPC3) were then transplanted subcutaneously into 8 Balb/c mice. In comparison, wild type 1MEA was also transplanted into 8 separate Balb/c mice. Further, we performed a comparative analysis of the gene expression of E-cadherin, vimentin, collagen I, fibronectin, twist, slug and snail in both wild type 1MEA and 1MEAhGPC3. 18 days post-transplantation, tumor volume was analyzed. 5 mice transplanted with wild type 1MEA had not yet developed tumors whereas 3 mice transplanted with 1MEAhGPC3 developed tumors that had grown past the allowed tumor volume for the protocol and were sacrificed. Analysis of the remaining mice (3 transplanted with 1MEA and 5 with 1MEAhGPC3 revealed that tumors from 1MEA were 8-fold more voluminous than tumors from 1MEAhGPC3. Comparative analysis of the gene expression profile of 1MEA versus 1MEAhGPC3 revealed that transfection with hGPC3 had resulted in increased expression of mesenchymal markers (collagen I by 27.5-fold, fibronectin by 18-fold, vimentin by 3-fold), loss of epithelial marker (E-cadherin loss by 99%) and increased expression of transcriptional regulators of EMT (twist by 11.7-fold, slug by 148-fold and snail by 8.7-fold).These data clearly show that hGPC3 promotes HCC progression by inducing EMT in HCC cells. To our knowledge, this is the first experimental evidence that hGPC3's role in HCC is associated with induction of EMT and it suggests that therapeutic targeting of GPC3 in human HCC may be an effective approach to the treatment of advanced HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2413. doi:1538-7445.AM2012-2413

Abstract 3429: Mesenchymal hepatocellular carcinoma cells acquire cancer stem cell characteristics through Snail1

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality and the incidence of HCC is rising in the United States. Cancer stem cells (CSCs) are a subset of neoplastic cells that possess distinct survival mechanisms and plasticity crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by Transforming Growth Factor-β (TGF-β) has been linked to the acquisition of CSC characteristics in breast cancer. In patients with HCC, either a CSC or EMT phenotype correlates with a worse prognosis. In this report, we aim to link EMT to CSCs and to further elucidate the mechanism by which cells acquire CSC characteristics during EMT. We utilized human (Huh7, MHCC97-L) and murine (P2E, P2M; Ding et al Hepatology. 2010) liver cancer cell lines, and analysis included gene and protein expression, FACS, migration and invasion assays, and chemotherapy resistance assay. Human mesenchymal MHCC97 cells demonstrate decreased E-cadherin, increased Snail1, and enhanced migration and invasion compared to epithelial Huh7 cells. MHCC97 cells demonstrate significant upregulation of stem cell markers (Oct4, Nanog, BMI-1, and CD44; p&amp;lt;0.01), increased CD44high population (95% vs. 2% Huh7, p&amp;lt;0.01), resistance to chemotherapy, resistance to TRAIL-induced apoptosis, increased tumor-sphere formation and slower cell cycle compared to epithelial Huh7 cells. We recently reported EMT in murine epithelial liver cell lines, P2E and P2M (post EMT). Mesenchymal P2M cells demonstrate loss of E-cadherin, gain of Snail1, increased tumor-sphere formation, and increased expression of stem cell markers (Nanog, Oct4, CD44, and EpCam; p&amp;lt;0.001). As TGF-β induces EMT through Snail1, we hypothesize that Snail1 signaling plays a role in the induction of an EMT and CSC phenotype in HCC. TGF-β treatment, epithelial P2E cells demonstrate fibroblastic-like morphology, 14-fold increase in Snail1 (p&amp;lt;0.01) and 8 fold increase in Nanog expression (p&amp;lt;0.01), increased tumor-sphere formation and migration. siRNA knockdown of Snail1 in mesenchymal P2M cells resulted in significant decrease in EMT and CSC characteristics, with decreased tumor-sphere formation, decreased migration, and decreased stem cell gene expression (Nanog and CD44, p&amp;lt;0.01). In conclusion, both murine and human mesenchymal HCC cells possess multiple CSC characteristics compared to epithelial counterparts. The transient knockdown of Snail1 in mesenchymal HCC cells results in the reversal of EMT as well as loss of CSC characteristics. We propose that Snail1 may play an important role in the regulation of EMT and CSC phenotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3429. doi:10.1158/1538-7445.AM2011-3429