- Email: [email protected]
1077 INTERLEUKIN 6, A POTENT REGULATOR OF Notch SIGNALING IN LIVER CANCER
POSTERS proteins, assessed with specific antibodies targeting histone H4 dimethylated on Lysine 20 (H4K20me2) and histone H4 acetylated on Lysine 16 (H4K16ac), was found significantly increased in HCC/MVI+ compared to HCC/MVI− in the train (p < 0.01) and validation (p < 0.05) cohorts. Conclusion: These results highlight the potential of MALDI IMS to uncover new relevant biomarkers of MVI in HCC. The use of these biomarkers would be helpful in the pre-operative management of patients with HCC. 1076 INTERLEUKIN 6 REGULATES LIVER CANCER STEM CELLS G. Wilson1 , J. George2 , A. Tian3 , Z. Hu3 , L. Qiao1 . 1 Medicine, University of Sydney, 2 Medicine, University of Sydney and Westmead Hospital, Westmead, NSW, Australia; 3 Gastroenterology, Lanzhou University First Hospital, Lanzhou, China E-mail: [email protected] Background and Aims: The majority of hepatocellular carcinoma (HCC) is caused by chronic hepatitis B and C virus infection. A proportion arises from cancer stem cells (CSCs) that are responsible for tumor initiation and progression. Interleukin 6 (IL-6), a pro-inflammatory cytokine involved in liver regeneration and the inflammatory response, is also thought to play a role in hepatocarcinogenesis, though the mechanisms are less clear. We sought to explore the regulatory role of IL-6 on hepatic CSCs. Methods: CD133+/CD44+ was used to isolate putative CSCs from HuH7 cells by FACScan and magnetic bead separation. Isolated CSCs were maintained in an undifferentiation state. The expression of commonly used CSC markers (Epcam, CD133, CD44, and AFP), stemness markers (Oct3/4, Nanog), as well as IL-6 signalling genes (IL-6, IL-6R, and STAT3) was examined by qPCR and Western blot. The tumour forming ability of the CD133+/CD44+ cells was examined by tumour sphere assays. Expression of CSC markers was also examined in mouse liver tissue up to 144 hours after administration of the carcinogen DEN. Results: Significantly higher level of IL-6, IL-6R, and pSTAT3 were observed in CD133+/CD44+ cells compared to CD133−/CD44− cells. CD133+/CD44+ cells demonstrated typical characteristics of CSCs, including enhanced expression of common CSC markers (CD133, CD44, Epcam, and AFP), stemness markers (Oct3/4, Nanog), and most importantly, an enhanced ability to form tumour spheres. In DEN-treated C57 mice, there was a significant up-regulation of IL-6 signalling at 24–48 h demonstrated by increased circulating IL-6, increased hepatic IL-6 and pSTAT3. These changes were accompanied by a temporospatial correlation of increases in the expression of CD44, Epcam, and AFP. Conclusions: IL-6 signalling is constitutively active in CSCs and may exert oncogenic effects through modulating stem cell stability. Studies are underway to investigate the impact of IL-6 signalling on the biological characteristics of hepatic CSCs. 1077 INTERLEUKIN 6, A POTENT REGULATOR OF Notch SIGNALING IN LIVER CANCER G. Wilson1 , J. George1 , Z. Hu2 , A. Tian3 , L. Qiao4 . 1 Medicine, University of Sydney, Westmead, NSW, Australia; 2 Gastroenterology, 3 Medicine, Lanzhou University First Hospital, Lanzhou, China; 4 Medicine, The University of Sydney, Westmead, NSW, Australia E-mail: [email protected] Background and Aims: Hepatocellular carcinoma (HCC) is an inflammation-mediated malignancy. Among the numerous proinflammatory cytokines, interleukin 6 (IL-6) plays a key role in driving carcinogenesis, but the mechanisms are not completely characterised. Notch signalling is a regulatory pathway that mediates cell differentiation, proliferation, apoptosis, and stem cell
maintenance. In this study we sought to explore the regulatory role of IL-6 on Notch signalling in liver cancer. Methods: HCC cell lines (HuH7, SNU182, SNU423) were transfected with an IL-6 over-expressing plasmid. In order to investigate the impact of IL-6 on Notch signalling, C57 and IL-6 knockout mice were injected with diethylnitrosamine (DEN) at 100 mg/kg and the livers were harvested. Quantitative real time PCR (qPCR) and Western blot were used to determine the expression of IL-6 and Notch signalling genes at the mRNA and protein levels. Stable HuH7 cells with IL-6 overexpression were injected into nude mice to test the oncogenic effect of IL-6. Results: In HCC cells, over-expression of IL-6 led to strong activation of IL-6 signalling, with increased IL-6 production and expression of IL-6 target genes (Jak1, Jak2, and pSTAT3). Overexpression of IL-6 led to increased cell proliferation and colony formation. These phenomena were reversed when cells were treated with the Notch inhibitor RO4929097 (Selleckchem). Livers from DEN treated C57 mice, revealed up-regulation of IL-6 and pSTAT3 up to 48 h. A temporospatial increase in the expression of Notch signalling genes (DLL4, Hes1, Hey1, NICD) was observed. In contrast, IL-6 KO mice did not respond to DEN with the above changes. Regression analysis of Western blot data demonstrated a correlation between IL-6 and Hes1, as well as IL-6 and DLL4 mRNA expression. In nude mice, IL-6 stable cell lines induced tumours that grew faster and at harvest were ~5-fold larger than xenografts from control cells. A significant increase in Notch target genes such as Hes1 and NICD was observed in IL-6 stable cell line-derived xenograft tumours. Conclusions: IL-6 may exert its oncogenic effect on liver, in part, through an up-regulation of Notch signalling. 1078 MECHANIC-BASED REPROGRAMMING OF HEPATIC SELF-RENEWING TUMOR-INITIATING CELLS THROUGH DNMT1-INHIBITION C. Raggi, V.M. Factor, S. Daekwan, G.C. Matthew, A. Holczbauer, J.U. Marquardt, S.S. Thorgeirsson. LEC, NCI, NIH, Bethesda, MD, USA E-mail: [email protected] Background and Aims: Modulation of cellular fate in solid tumors is defined to a large extent by DNMT1-regulated epigenetic machinery and cellular-non-cellular constituents in the tumorinitiating cell (TIC) niche. Current study examines the significance of the DNMT1-cellular interactions in reprogramming of TICs properties. Methods: Seven HCC cell lines were plated in 2D culture at various cell densities and exposed to a transient nontoxic dose of a DNMT1-inhibitor Zebularine (ZEB). After a 3-day treatment, cells were cultured in 3D non-adherent condition in ZEB- and serumfree media to generate primary spheres (G1) which were then passaged through generation G5. Differences in long-term selfrenewal, gene expression, tumorigenicity and metastatic potential of G1-G5 spheres were examined. Results: Transient exposure to ZEB produced the differential cell density-depended responses in 5/7 tested HCC cell lines. In cells grown at low density (LD), ZEB caused a remarkable increase in G1 sphere formation. This effect persisted through G5. In striking contrast, untreated LD cells failed to form primary spheres while the sphere forming potential of high density (HD) and HD ZEB-treated (HDZ) cells rapidly decreased over the first 3 generations. Likewise DNMT1 depletion by shRNA promoted acquisition of self-renewal potential in LD cells. The increase in sphere forming potential of LDZ cells strongly correlated with a stable overexpression of cancer stem cell-related markers and key genes involved in self-renewal and epithelial–mesenchymal transition. Moreover, when dissociated LDZ, HD and HDZ spheres were injected subcutaneously into NOD/SCID mice, LDZ cells generated tumors more rapidly and were more metastatic. Both
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maintenance. In this study we sought to explore the regulatory role of IL-6 on Notch signalling in liver cancer. Methods: HCC cell lines (HuH7, SNU182, SNU423) were transfected with an IL-6 over-expressing plasmid. In order to investigate the impact of IL-6 on Notch signalling, C57 and IL-6 knockout mice were injected with diethylnitrosamine (DEN) at 100 mg/kg and the livers were harvested. Quantitative real time PCR (qPCR) and Western blot were used to determine the expression of IL-6 and Notch signalling genes at the mRNA and protein levels. Stable HuH7 cells with IL-6 overexpression were injected into nude mice to test the oncogenic effect of IL-6. Results: In HCC cells, over-expression of IL-6 led to strong activation of IL-6 signalling, with increased IL-6 production and expression of IL-6 target genes (Jak1, Jak2, and pSTAT3). Overexpression of IL-6 led to increased cell proliferation and colony formation. These phenomena were reversed when cells were treated with the Notch inhibitor RO4929097 (Selleckchem). Livers from DEN treated C57 mice, revealed up-regulation of IL-6 and pSTAT3 up to 48 h. A temporospatial increase in the expression of Notch signalling genes (DLL4, Hes1, Hey1, NICD) was observed. In contrast, IL-6 KO mice did not respond to DEN with the above changes. Regression analysis of Western blot data demonstrated a correlation between IL-6 and Hes1, as well as IL-6 and DLL4 mRNA expression. In nude mice, IL-6 stable cell lines induced tumours that grew faster and at harvest were ~5-fold larger than xenografts from control cells. A significant increase in Notch target genes such as Hes1 and NICD was observed in IL-6 stable cell line-derived xenograft tumours. Conclusions: IL-6 may exert its oncogenic effect on liver, in part, through an up-regulation of Notch signalling. 1078 MECHANIC-BASED REPROGRAMMING OF HEPATIC SELF-RENEWING TUMOR-INITIATING CELLS THROUGH DNMT1-INHIBITION C. Raggi, V.M. Factor, S. Daekwan, G.C. Matthew, A. Holczbauer, J.U. Marquardt, S.S. Thorgeirsson. LEC, NCI, NIH, Bethesda, MD, USA E-mail: [email protected] Background and Aims: Modulation of cellular fate in solid tumors is defined to a large extent by DNMT1-regulated epigenetic machinery and cellular-non-cellular constituents in the tumorinitiating cell (TIC) niche. Current study examines the significance of the DNMT1-cellular interactions in reprogramming of TICs properties. Methods: Seven HCC cell lines were plated in 2D culture at various cell densities and exposed to a transient nontoxic dose of a DNMT1-inhibitor Zebularine (ZEB). After a 3-day treatment, cells were cultured in 3D non-adherent condition in ZEB- and serumfree media to generate primary spheres (G1) which were then passaged through generation G5. Differences in long-term selfrenewal, gene expression, tumorigenicity and metastatic potential of G1-G5 spheres were examined. Results: Transient exposure to ZEB produced the differential cell density-depended responses in 5/7 tested HCC cell lines. In cells grown at low density (LD), ZEB caused a remarkable increase in G1 sphere formation. This effect persisted through G5. In striking contrast, untreated LD cells failed to form primary spheres while the sphere forming potential of high density (HD) and HD ZEB-treated (HDZ) cells rapidly decreased over the first 3 generations. Likewise DNMT1 depletion by shRNA promoted acquisition of self-renewal potential in LD cells. The increase in sphere forming potential of LDZ cells strongly correlated with a stable overexpression of cancer stem cell-related markers and key genes involved in self-renewal and epithelial–mesenchymal transition. Moreover, when dissociated LDZ, HD and HDZ spheres were injected subcutaneously into NOD/SCID mice, LDZ cells generated tumors more rapidly and were more metastatic. Both
Journal of Hepatology 2013 vol. 58 | S409–S566
S441