Page 109 - ebook HCC

GENEVA, SWITZERLANDA, SWITZERLAND
PROGRAMME
EASL
106 PROGRAMME AND ABSTRACTSAND ABSTRACTS GENEV EASL HCC SUMMITHCC SUMMIT 107
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107
FEBRUARY 13 - 16, 2014Y 13 - 16, 2014
FEBRUAR
Poster Board Number B9 Poster Board Number B10
NUP155 IS REQUIRED FOR FULL INDUCTION OF HYPOXIA INDUCES AN INCREASED NUMBER OF
A SUBSET OF P53 TARGET GENES IN HCC PROGENITOR CELLS IN LATE BUT NOT IN EARLY
STAGES OF HEPATOCELLULAR CARCINOMA

Kerstin Holzer , Alessandro Ori , Juliane Winkler , Eva Eiteneuer , Martin Beck ,
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Peter Schirmacher , Stephan Singer 1 2 Eliene Bogaerts , Femke Heindryckx , Anja Van den Bussche ,
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1 Institute of Pathology, University Hospital Heidelberg, 69120 Heidelberg, 1 1
Anja Geerts , Hans Van Vlierberghe
2 Structural and Computational Biology, European Molecular Biology Laboratory, 1 Gastro-enterology and hepatology, Ghent university, Ghent, Belgium, Department of
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69117 Heidelberg, Germany Medical Biochemistry and Microbiology, Uppsala university, Uppsala, Sweden
Corresponding author’s e-mail: kerstin.holzer@med.uni-heidelberg.de Corresponding author’s e-mail: eliene.bogaerts@gmail.com
Introduction: The nuclear pore complex (NPC) consists of approximately 30 different Introduction: Hepatocellular carcinoma(HCC) has a high incidence and is often detected
nucleoporins (Nups) and is embedded in the nuclear envelope. Almost all signaling in advanced stages. Current treatment options are mostly based on nutrient deprivation by
cascades of oncogenic and tumorsuppressive pathways have to pass the NPC representing decreasing angiogenesis, thus creating hypoxic conditions. This could lead to an altered
the only gate between the cytoplasm and the nucleoplasm. Recent data suggest that NPC liver progenitor cell (LPC)-niche, creating a more aggressive, resistant tumor phenotype.
components can modulate cancer-relevant pathways on different levels. Understanding more about the influence of hypoxia on LPCs, is of vast importance to
improve hypoxia inducing therapies.
Aims: We aim to define the role of Nup155 in modulating the p53 pathway in HCC.
Aims: Compare the effect of hypoxic conditions at different time points in
hepatocarcinogenesis to determine the time-dependent effect of hypoxia on LPC activation.
Methodology: In HepG2 cells (p53 wild-type) Nup155 was depleted by using two different
BASIC POSTER ABSTRACTS experiments were performed in Hep3B-4Bv cells harboring a temperature-sensitive p53 Methodology: HCC was induced in mice by weekly diethylnitrosamine (DEN) injections BASIC POSTER ABSTRACTS
siRNAs followed by Nutlin- or Camptothecin(CPT) treatment to induce p53. Similar
(35mg/kg) for 22 weeks. To mimic a hypoxic reaction, pan-prolyl-hydroxylase-domain
mutant that acquires wild-type function by shifting the incubation temperature from 37° to
inhibitor dimethyloxaloylglycine (DMOG) was administered twice a week (300µL,16mg/mL)
32°. The impact of Nup155 knockdown on p53 target gene expression was investigated
for 5weeks from week16-22, or therapeutically from week 22-27. Number of cytokeratin 19
on protein and mRNA level by immunoblotting and qRT-PCR, respectively. Subsequent
(CK19) positive singular (LPCs) and ductular (cholangiocytes) cells per portal region were
mechanistic analyses involved p21 protein half-life experiments by using cycloheximide
treatment blocking mRNA translation. Potential alterations of p21 mRNA export were
CK 7 and 19 were evaluated through qPCR analysis
tested by subcellular fractionation followed by qRT-PCR. To identify other p53 targets with
Nup155-dependency and to analyze global protein expression changes in the presence or counted and mRNA levels of the LPC markers prominin1(CD133), epithelial cadherin and
absence of Nup155 we conducted shotgun proteomics. Results: Both DMOG and PBS from week 16 to 22 in DEN induced mice resulted in
significantly increased numbers of ductular and singular cells compared to saline control.
Results: Depletion of Nup155 in HepG2 cells by RNAi was followed by a significantly Interestingly, on the RNA level, DMOG from week 16 to 22 only caused upregulation of
reduced p21 protein accumulation upon Nutlin- and CPT-treatment. A selective impact on CD133, while in the PBS group, all LPC markers were significantly upregulated compared
p21 induction after Nup155 knockdown was also observed in Hep3B-4Bv cells and in a cell to saline control. Even though no significant differences could be marked between PBS
line expressing p21 from a tet-sensitive cDNA construct (“tet-off”). Interestingly, p21 mRNA and DMOG groups, increased stabilisation of the hypoxia inducible factor seems to play a
induction was not significantly affected in these cells. Cycloheximide chase experiments did negative role in LPC activation and proliferation at this early stage.
not indicate obvious changes of p21 protein half-life upon Nup155 depletion. Furthermore, Alternatively, therapeutically induced DMOG resulted in a significant increase in ductular
p21 mRNA export was not significantly altered by Nup155 knockdown indicated by an and singular CK19+ cells compared to both PBS and saline control. After 22 weeks of
unchanged nuclear/cytoplasmic p21 mRNA-ratio. Hypothesizing that Nup155 may affect p21 DEN, 5w of PBS treatment showed no increase in the number of LPC or ducts in portal
mRNA translation we are currently performing polysomal fractionation. Shotgun proteomics areas, qPCR analysis did show CK19 and CD133 significantly upregulated compared to
revealed distinct changes of protein expression after Nup155 depletion with the cytoplasmic saline control. In the therapeutic DMOG group , mRNA levels of all LPC markers were
FMR1 interacting protein 2 (CYFIP2) being another p53 target dependent on Nup155. significantly increased compared to saline control and CK7 compared to PBS.
Conclusions: We show timing to be essential in hypoxia mediated LPC activation and
Conclusions: We conclude that Nup155 regulates a subset of p53 target genes on a
post-transcriptional level by a different mechanism than previously described for Nup98. differentiation in HCC. Possibly the lack of LPC response in earlier stages could unveil a
safe window for hypoxia inducing treatments.