DISSERTATION Titel der Dissertation „Novel Role of STAT3 in Hepatocellular Carcinogenesis“ Band 1 von 1 Verfasser Mag.rer.nat. Georg Machat angestrebter akademischer Grad Doktor der Naturwissenschaften (Dr.rer.nat.) Wien, 2013 Studienkennzahl lt. Studienblatt: A 091 441 Dissertationsgebiet lt. Studienblatt: Genetik und Mikrobiologie Betreuerin / Betreuer: Ao. Univ. Prof. Mag. Dr. Wolfgang Mikulits Table of contents: 1 Abstract .........................................................................................................................1 2 Zusammenfassung ........................................................................................................2 3 Introduction ..................................................................................................................3 3.1 Hepatocellular Carcinoma ........................................................................................3 3.2 p14ARF/p19ARF ........................................................................................................ 14 3.2.1 The ARF-p53 Pathway .................................................................................... 14 3.2.2 p19ARF/p14ARF p53-Independent Tumor Suppression ....................................... 16 3.2.3 p19ARF/p14ARF in HCC .................................................................................... 20 3.3 JAK-STAT Pathway and STAT3 ............................................................................ 21 3.3.1 JAK-STAT ...................................................................................................... 21 3.3.2 The Structure and Function of STAT Proteins ................................................. 22 3.3.3 STAT3 ............................................................................................................ 24 3.4. Aims of the study ....................................................................................................... 35 4 Manuscript .................................................................................................................. 36 4.1 Contribution to this study ....................................................................................... 37 4.2 Abstract .................................................................................................................. 38 4.3 Introduction ............................................................................................................ 39 4.4 Materials and Methods ........................................................................................... 41 4.5 Results ................................................................................................................... 43 4.5.1 Stat3 represses tumor growth of Ras-transformed p19ARF-/- hepatocytes ........... 43 4.5.2 Loss of Stat3 promotes tumor formation in p19ARF-/- MIM-R hepatocytes ........ 43 4.5.3 Stat3 acts pro-oncogenic in p19ARF-positive Ras-transformed hepatocytes ....... 44 4.5.4 Upregulation of p19ARF is associated with DEN-induced tumor formation in Stat3fl/fl mice.................................................................................................... 44 4.5.5 p14ARF modulates Stat3 activation during human HCC development ............... 45 4.5.6 p14ARF acts downstream of Jak-mediated Stat3 phosphorylation ...................... 45 4.6 Discussion .............................................................................................................. 47 4.7 References .............................................................................................................. 50 4.8 Figures ................................................................................................................... 53 4.9 Supplemetary data .................................................................................................. 60 4.9.1 Supporting Figures .......................................................................................... 60 4.9.2 Supporting Material and Methods .................................................................... 63 5 Results ......................................................................................................................... 65 5.1 Microarray analysis of murine STAT3-deficient HCC cells expressing STAT3 isoforms ................................................................................................................. 65 5.2 Murine HCC cells show functional p53 pathway .................................................... 65 5.3 Unphosphorylated STAT3 (U-STAT3) translocates to the nucleus and is transcriptionally active ........................................................................................... 66 5.4 NFkB translocates to the nucleus irrespective of U-STAT3 .................................... 67 5.5 Suppression of STAT3 phosphorylation in p14ARF knockdown Hep3B cells occurs early in tumor development .................................................................................... 68 5.6 Proliferation of Hep3B cells lacking p14ARF in vitro is independent of STAT3 activation ................................................................................................................ 71 5.7 Exogenous expression of p14ARF leads to decreased tumor formation and vascularization ....................................................................................................... 71 5.8 The impact of STAT3 and/or p14 knockdown is cell line dependent ....................... 74 5.8.1 Knockdown of STAT3 in human Hep3B hepatoma cells ................................. 74 5.8.2 Hep3B-shSTAT3-shp14 .................................................................................. 75 5.8.3 PLC-shSTAT3 ................................................................................................ 76 5.9 Detection of p14ARFin primary human HCC............................................................ 77 5.10 The TGF-β-Smad pathway is crucial in murine, but dispensable in human hepatoma cells .................................................................................................... 78 5.11 Phosphorylation of STAT3 is independent of PTEN ........................................... 79 6 Discussion .................................................................................................................... 82 6.1 p19ARF/p14ARF Controls Oncogenic Functions of STAT3 in HCC – in retrospect .... 82 6.2 Further investigations of murine model systems ..................................................... 85 6.2.1 Microarray analysis ......................................................................................... 85 6.2.2 p53 functionality ............................................................................................. 86 6.2.3 U-STAT3 localization and transactivation ....................................................... 86 6.2.4 The interaction of NFkB and U-STAT3 ........................................................... 87 6.2.5 Conclusions and outlook ................................................................................. 87 6.3 Investigation on human hepatoma cell lines – facing diversity ................................ 88 6.3.1 Early down-regulation of active STAT3 in Hep3B-shp14ARF cells ................ 88 6.3.2 Impact of p14ARF expression ............................................................................ 89 6.3.3 Intervention with STAT3 ................................................................................. 90 6.3.4 Double knockdown of STAT3/p14ARF in human hepatoma cells ................... 90 6.3.5 Analysis of p14ARF in primary human HCC .................................................. 91 6.3.6 The role of STAT3 in TGF-β signaling ............................................................ 92 6.3.7 Investigations on PTEN/STAT3 interactions in human HCC ........................... 93 6.4 Concluding remarks ............................................................................................... 95 7 References ................................................................................................................... 98 8 Materials and Methods ............................................................................................. 119 9 Acknowledgements ................................................................................................... 125 10 Curriculum Vitae ...................................................................................................... 126 Dissertation Georg Machat 1 Abstract Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. Chronic liver disease caused by viral hepatitis infection, steatohepatitis or intoxication by Aflatoxin or alcohol represents the main background for HCC development. Deregulation of various signaling cascades such as aberrations in Ras and STAT (signal transducer and activator of transcription) signaling generates heterogeneous molecular patterns of HCC. This study addressed the role of STAT3 in HCC. Albeit known as an oncogene in HCC, STAT3 showed both pro- and anti-oncogenic features in Ras-transformed murine hepatoma cells which are under the control of p19ARF. Knockout of STAT3 as well as exogenous expression of STAT3 lacking the phosphorylation site on Tyr705 (U-STAT3) caused enhanced tumor formation, demonstrating a tumor-suppressive function of STAT3 in Ras-transformed hepatocytes that are deficient for p19ARF. Furthermore, the knockout of STAT3 abrogated the anti-proliferative