Supplementary Data

Materials and methods

Cell culture

HepG2 human hepatoblastoma cells were obtained from ATCC (HB-8065,

Rockville, MD, USA). Cells were grown in a humidified incubator (20% O2, 5%

CO2 at 37°C) (Sanyo MCO-18M O2/CO2 incubator, Osaka, Japan) in Williams

Medium E (WEM, InVitrogen) supplemented with 10% fetal calf serum, 2 mM L-

glutamine, 20 mU/ml insulin, 50 nM dexamethasone, 100 U/ml penicillin, 100

µg/ml streptomycin, 2.5 µg fungizone, 50 µg/ml gentamycin and 100 µg/ml vancomycin (=WEM-C).

For the microarray analysis two experiments were executed in parallel. Cells

6 were seeded at 3x10 in 75 cm² tissue culture flasks (n=4) at 20% O2 and were

grown until 70% confluence. After reaching near-confluence, 2 flasks were

placed in a humidified incubator with hypoxic conditions (2% O2, 5% CO2 at

37°C), while the other flasks (n=2) remained in normoxic conditions (20% O2).

Cells were cultured for 72hrs in these different oxygen conditions and after three days cells were harvested after trypsin treatment, mixed with Trizol (InVitrogen,

Merelbeke, Belgium) and stored in -80°C for further analysis.

Sample Collection and Microarray Target Synthesis and Processing

Samples in Trizol were homogenized in a Dounce homogenizer for RNA extraction. Thereafter, RNA was isolated with the RNeasy Kit (Qiagen,

Chatsworth, CA) according to the manufacturer’s instructions. The quality of all RNA samples was monitored with a NanoDrop spectrophotometer (NanoDrop

Technologies, Centreville, DE) and by means of the Agilent 2100 BioAnalyzer

(Agilent, Palo Alto, CA). Only RNA showing no signs of degradation or impurities

(260/280 and 260/230 nm ratios, >1.8) was considered suitable for microarray

analysis and used for labeling. Briefly, from 1 μg of cellular RNA, poly-A RNA

was reversed transcribed using a poly dT-T7 primer. The resulting cDNA was

immediately used for one round of amplification by T7 in vitro transcription

reaction in the presence of Cyanine 3-CTP or Cyanine 5-CTP. The amplified and

labeled RNA probes were purified separately with RNeasy purification columns

(Qiagen, Belgium). Probes were verified for amplification yield and incorporation

efficiency by measuring the RNA concentration at 280 nm, Cy3 incorporation at

550 nm and Cy5 incorporation at 650 nm using a Nanodrop spectrophotometer.

Samples were hybridized on dual color Agilent's Human Whole Genome Oligo

Microarray (Cat# G4112F, Agilent, Diegem, Belgium) that contained 44k 60-mer

oligonucleotide probes representing around 41,000 well-characterized human

transcripts. Agilent technology utilizes one glass array for the simultaneous

hybridization of two populations of labeled, antisense cRNAs obtained from two samples (reference and assay).

Primary data analysis

Statistical data analysis was performed on the processed Cy3 and Cy5 intensities, as provided by the Feature Extraction Software version 9.1. Probes with none of the eight signals flagged as positive and significant (by the Feature Extraction Software) were omitted from all subsequent analyses as well as the

various controls. Further analysis was performed in the R programming

environment, in conjunction with the packages developed within the Bioconductor project (http://www.bioconductor.org).(1) In a first analysis the differential

expression of the 2% versus 20% oxygen samples was assessed via the

moderated t-statistic, described in Smyth (2004).(2) This moderated statistic

applies an empirical Bayesian strategy to compute the gene-wise residual

standard deviations and thereby increases the power of the test, especially

beneficial for smaller data sets. To control the false discovery rate, multiple

testing correction was performed and probes with a corrected p-value below 0.05

and a fold change of >2 were selected.(3) To determine the highly significant

differentially expressed genes under chronic hypoxic conditions we used higher stringency of p-value below 0.01. Finally, the remaining differentially expressed genes were designated as the liver in vitro hypoxia gene set and with these genes we could further investigate the relevance of chronic hypoxia in primary human liver cancer.

Quantitative RT-PCR

- Hypoxia score at different oxygen tension

To investigate the expression of the 7 genes from the in vitro hypoxia gene set at different oxygen concentrations a number of parallel experiments were performed. HepG2 cells were seeded in 25cm³ culture flasks (106 cells/flask) that were placed in either reduced O2 (5% O2, 2% O2 and 1%O2) or 20% O2 in parallel. All culture conditions were performed in triplicate and cells were

collected for RNA isolation. Samples were processed for RT-PCR as described

below (for primers see Table 1A).

- Confirmation array results

To investigate the dynamics of hypoxia related gene expression and to confirm

the array findings, we performed RT-PCR at different time points for an additional set of 6 genes (Table 1B) using beta-2-microglobulin as housekeeping gene.

HepG2 cells were seeded in 25cm³ culture flasks (106 cells/flask), using the

same culture conditions as were used for the microarray experiment. Flasks were

placed in either 2% O2 or 20% O2 and gene expression was tested at 0 hr, 10

hrs, 24 hrs and up to 72 hrs. All culture conditions were performed in triplicate

and cells were collected for RNA isolation.

RNA was isolated with the RNeasy Kit (Qiagen, Chatsworth, CA) according to

the manufacturer’s instructions. One microgram of cellular RNA was reverse

transcribed into cDNA using SuperScript II reverse transcriptase and random

hexamer primers (Invitrogen Life Technologies, USA).

The PCR reaction was carried out in a volume of 10 µl in a mixture that contained

appropriate sense- and anti-sense primers and a probe in TaqMan Universal

PCR Master Mixture (Applied Biosystems, Foster City, California). We used the

Assays-on-DemandTM Gene Expression products, which consist of a 20x mix of

unlabeled PCR primers and TaqMan MGB probe (FAMTM dye-labeled). These assays are designed for the detection and quantification of specific human genetic sequences in RNA samples converted to cDNA. The primers used are listed in Table 1.

Real-time PCR amplification and data analysis were performed using the A7500

Fast Real-Time PCR System (Applied Biosystems). Each sample was assayed in duplicate in a MicroAmp optical 96-well plate. The thermo-cycling condition consisted of 2 minutes at 50°C and 10 min incubations at 95°C, followed by 40 two-temperature cycles of 15 seconds at 95°C and 1 min at 60°C. The ΔΔCt- method was used to determine relative gene expression levels.

Gene Gene Name Chromosome Assay ID Applied symbol Biosystems A Hypoxia score at different oxygen tension B2M Beta-2-microglobulin 15 Hs99999907_m1 CCNG2 cyclin G2 4 Hs00171119_m1 EGLN3 egl nine homolog 3 (C. elegans) 14 Hs00222966_m1 ERO1L ERO1-like (S. cerevisiae) 14 Hs00969232_m1 FGF21 fibroblast growth factor 21 19 Hs00173927_m1 MAT1A methionine adenosyltransferase I, alpha 10 Hs01547962_m1 RCL1 RNA terminal phosphate cyclase-like 1 9 Hs00195050_m1 WDR45L WDR45-like 17 Hs00750495_s1 B Confirmation array results

CDO1 Cysteine dioxygenase, type I 5 Hs00156447_m1 EGLN1 Egl nine homolog 1 (C. elegans) 1 Hs00254392_m1 HIF-1α Hypoxia-inducible factor 1, alpha subunit 14 Hs00936368_m1 FIH Hypoxia-inducible factor 1 alpha inhibitor 10 Hs00215495_m1 IGFBP3 Insulin-like growth factor binding protein 3 7 Hs00181211_m1 VEGF-A Vascular endothelial growth factor A 6 Hs00173626_m1

Table 1. List of genes and RT-PCR assay IDs (Applied Biosystems) used in this study. Immunohistochemistry on Hif-1α and VEGF

HepG2 cells were grown on Thermanox plastic cover slips (Nalgene Nunc

international, Rochester, NY USA, 13 mm diameter) placed in a 24 well plate with

1 mL William’s Medium E (WEM-C, InVitrogen). After one day of incubation and

attachment, cells were either exposed to hypoxia (2% O2) or normal oxygen

conditions for 0, 24, or 72 hours. Subsequently cells were washed once with PBS

and fixed in acetone for 15 minutes. When dry, the cover slides were stored at -

20°C.

For immunohistochemistry we used the Envision technique of Dako. Cover slips

collected at the different time points were stained in duplicate. Cells were

incubated for 45 minutes with a primary antibody against Hif-1α (1:250 anti-Hif-

1α monoclonal mouse antibody, BD Biosciences) or against VEGF (1:100 anti-

VEGF A-20 polyclonal rabbit antibody, Santa Cruz). As secondary antibody

Envision monoclonal antibodies were used (for Hif-1α; Envision monoclonal mouse antibody, Dako and for VEGF; Envision monoclonal rabbit antibody,

Dako). Finally, the staining was performed with 3-amino-9-ethylcarbazole (AEC)

and a contra-staining with haematoxylin. The thermanox cover slips were

mounted with glycergel. To evaluate the staining we used a semi-quantative

quickscore,(4) which combines positivity (P) and intensity (I). Positivity was

scored as: 1= 0-4%, 2= 5-19%, 3= 20-39%, 4= 40-59%, 5= 60-79% and 6= 80-

100%. Intensity was scored as: 0= negative, 1= weak, 2= intermediate and 3= strong. The final score was the total of P+I and has a range of 1-9. All slides were scored independently by two researchers.

Results

Microarray

We started with the cell culture as model and determined the differentially expressed genes in HepG2 cells that were cultured for 72 hours at either 20% oxygen or in hypoxic conditions at 2% oxygen. We used the Agilent technology with color flip on two independent experiments in duplicate resulting in 8 ratio values. A total of 37,707 spots showed a representative signal of which 3,592

(8%) with a fold change above 2 and an uncorrected p-value <0.05 (1,879 up- regulated and 1,713 down-regulated). To control the false discovery rate, multiple testing correction was performed and the highly significant genes with a corrected p-value below 0.01 and a fold change of >2 were selected. This resulted in the identification of 265 genes (207 upregulated and 58 downregulated) designated as the hypoxic gene set (available as separate pdf- file, Table 2). Microarray data are also available at NCBI under number

GSE15366.

Table 2. Hypoxic gene set. The table provides a list of the differentially expressed genes (fold change above 2 and Limma correction p<0.01) in

cultures of HepG2 cells exposed to hypoxia (2% O2) for 72 hours compared to

cells grown at 20% O2.

Fold change ProbeName GeneSymbol GeneName log-ratio(2%/20%) UP A_23_P159325 ANGPTL4 angiopoietin-like 4 5.372466499 A_23_P312150 EDN2 endothelin 2 5.180316667 A_23_P170719 5.127389855 A_23_P118854 KRTHA7 keratin, hair, acidic, 7 4.535917168 A_23_P408249 PCK1 phosphoenolpyruvate carboxykinase 1 (soluble) 4.445758677 A_23_P122216 LOX lysyl oxidase 4.368888976 A_24_P565496 4.190445219 A_24_P565503 4.096483848 A_23_P216966 PTGS1 prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) 4.038067459 A_24_P220485 OLFML2A olfactomedin-like 2A 3.915798485 A_23_P62752 NPPB natriuretic peptide precursor B 3.91034339 A_23_P56949 SERTAD2 SERTA domain containing 2 3.78484135 A_23_P148473 IL2RG interleukin 2 receptor, gamma (severe combined immunodeficiency) 3.773126687 A_23_P127948 ADM adrenomedullin 3.729124757 A_24_P320699 IGFBP3 insulin-like growth factor binding protein 3 3.708987369 A_32_P29140 EST50565 Gall bladder I Homo sapiens cDNA 5- end, mRNA sequence 3.669404985 A_23_P26124 RORA RAR-related orphan receptor A 3.664936375 A_23_P374689 GAD1 glutamate decarboxylase 1 (brain, 67kDa) 3.658133484 A_24_P655888 3.616480556 A_23_P121885 ROPN1L ropporin 1-like 3.520250354 A_23_P64721 GPR109B G protein-coupled receptor 109B 3.516799888 A_23_P360379 EGLN3 egl nine homolog 3 (C. elegans) 3.505047745 A_24_P362904 PFKFB4 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 3.49982594 A_23_P83579 ARNT2 aryl-hydrocarbon receptor nuclear translocator 2 3.428158577 A_32_P218707 3.418748145 A_24_P80204 MALL mal, T-cell differentiation protein-like 3.379124085 A_23_P111995 LOXL2 lysyl oxidase-like 2 3.357153762 A_23_P6433 MB myoglobin 3.328240169 A_23_P218774 RAC2 ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2) 3.317676718 A_24_P38387 NDRG1 N-myc downstream regulated gene 1 3.226983198 A_23_P163666 WFIKKN1 WAP, follistatin/kazal, immunoglobulin, kunitz and netrin domain containing 1 3.196971634 A_23_P341938 NOG noggin 3.167625766 A_23_P205370 ASB2 ankyrin repeat and SOCS box-containing 2 3.122253906 A_24_P237586 ANKRD37 ankyrin repeat domain 37 3.119026953 A_23_P420863 CARD15 caspase recruitment domain family, member 15 3.100916452 A_23_P107454 KRTAP3-1 keratin associated protein 3-1 3.077494053 A_24_P236091 ENO2 enolase 2 (gamma, neuronal) 3.063400002 A_23_P119143 ICAM5 intercellular adhesion molecule 5, telencephalin 3.056328601 A_23_P83339 RNF183 ring finger protein 183 3.053588243 A_23_P11629 TMEM61 transmembrane protein 61 2.979491974 A_23_P102454 INSIG2 insulin induced gene 2 2.976653058 A_23_P141802 SERPINB7 serpin peptidase inhibitor, clade B (ovalbumin), member 7 2.973886054 A_23_P110266 FLJ23191 hypothetical protein FLJ23191 2.958333193 A_23_P119478 EBI3 Epstein-Barr virus induced gene 3 2.950487054 A_23_P397978 FLJ40722 hypothetical protein FLJ40722 2.932467673 A_23_P123234 2.926170585 A_32_P46238 LOC339240 keratin pseudogene 2.915716785 A_32_P47754 SLC2A14 solute carrier family 2 (facilitated glucose transporter), member 14 2.885480816 A_23_P161399 MXI1 MAX interactor 1 2.86641065 A_23_P79398 IL1R2 interleukin 1 receptor, type II 2.798884071 A_23_P120863 GAL3ST1 galactose-3-O-sulfotransferase 1 2.77297059 A_24_P251599 CAV3 caveolin 3 2.749002486 A_23_P204947 GJB2 gap junction protein, beta 2, 26kDa (connexin 26) 2.747346884 A_23_P88849 RRAD Ras-related associated with diabetes 2.736246118 A_23_P120504 C20orf46 chromosome 20 open reading frame 46 2.714119905 A_23_P413788 FUT11 fucosyltransferase 11 (alpha (1,3) fucosyltransferase) 2.713561269 A_23_P77389 2.696530787 A_23_P76071 B3GNT4 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 4 2.689241688 A_23_P119502 EDG6 endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 6 2.673194268 A_23_P150379 EVA1 epithelial V-like antigen 1 2.652006737 A_23_P370651 FAM13A1 family with sequence similarity 13, member A1 2.644533618 A_32_P132317 GPR155 G protein-coupled receptor 155 2.635247712 A_24_P81900 SLC2A3 solute carrier family 2 (facilitated glucose transporter), member 3 2.624644577 A_23_P16469 PLAUR plasminogen activator, urokinase receptor 2.609647791 A_23_P32474 CDCP1 CUB domain containing protein 1 2.608542254 A_23_P111888 CTHRC1 collagen triple helix repeat containing 1 2.588459925 A_23_P113212 TMEM45A transmembrane protein 45A 2.585041097 A_23_P142345 PRTN3 proteinase 3 (serine proteinase, neutrophil, Wegener granulomatosis autoantigen) 2.563793905 A_23_P157793 CA9 carbonic anhydrase IX 2.559319876 A_24_P37903 2.551683616 A_24_P98948 2.529267335 A_23_P71880 SPINK4 serine peptidase inhibitor, Kazal type 4 2.517922968 A_23_P166087 RASSF2 Ras association (RalGDS/AF-6) domain family 2 2.510356392 A_24_P378019 IRF7 interferon regulatory factor 7 2.507510643 A_23_P60627 ALOX15B arachidonate 15-lipoxygenase, type B 2.478244747 A_24_P229389 DKFZp686I15217 hypothetical protein DKFZp686I15217 2.466167917 A_24_P201702 CLEC2B C-type lectin domain family 2, member B 2.454926584 A_23_P95417 TNFRSF10D tumor necrosis factor receptor superfamily, member 10d, decoy with truncated death domain 2.454712156 A_23_P49448 FA2H fatty acid 2-hydroxylase 2.454016448 A_24_P414553 ARRDC3 arrestin domain containing 3 2.453536035 A_23_P308483 MAP3K15 mitogen-activated protein kinase kinase kinase 15 2.443747969 A_24_P406693 P4HA1 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide I 2.431465879 A_23_P211267 RIPK4 receptor-interacting serine-threonine kinase 4 2.430933708 A_24_P373976 SDC4 syndecan 4 (amphiglycan, ryudocan) 2.418633958 A_23_P132027 SPAG4 sperm associated antigen 4 2.407799543 A_23_P76480 CLEC2B C-type lectin domain family 2, member B 2.396611522 A_23_P201538 JUN v-jun sarcoma virus 17 oncogene homolog (avian) 2.383489201 A_24_P158089 SERPINE1 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 2.370217952 A_23_P426501 TLE6 transducin-like enhancer of split 6 (E(sp1) homolog, Drosophila) 2.369791626 A_32_P83845 HEY1 hairy/enhancer-of-split related with YRPW motif 1 2.369258616 A_24_P379104 PIM2 pim-2 oncogene 2.361586276 A_32_P627 FLJ23867 hypothetical protein FLJ23867 2.354789613 A_23_P303833 SCN4B sodium channel, voltage-gated, type IV, beta 2.348403477 A_24_P407311 ERO1L ERO1-like (S. cerevisiae) 2.335888455 A_23_P57836 2.332981055 A_24_P339944 PDGFB platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog) 2.331767624 A_23_P113005 EFNA1 ephrin-A1 2.331019252 A_23_P144476 SPRY1 sprouty homolog 1, antagonist of FGF signaling (Drosophila) 2.330819287 A_23_P143016 ARID5A AT rich interactive domain 5A (MRF1-like) 2.329222678 A_23_P205531 RNASE4 ribonuclease, RNase A family, 4 2.325029085 A_23_P51376 FAM77C family with sequence similarity 77, member C 2.32401213 A_23_P211910 PLOD2 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 2.31740339 A_23_P216693 MLLT3 myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 3 2.31384484 A_24_P942163 FAM63A family with sequence similarity 63, member A 2.281598558 A_23_P66347 2.273795017 A_24_P221551 MICAL3 Microtubule associated monoxygenase, calponin and LIM domain containing 3 2.25091082 A_23_P501933 CACNG6 calcium channel, voltage-dependent, gamma subunit 6 2.247456734 A_23_P256542 C3orf28 chromsome 3 open reading frame 28 2.244070287 A_24_P54670 SLC6A6 solute carrier family 6 (neurotransmitter transporter, taurine), member 6 2.241149276 A_23_P408708 BNIP3L BCL2/adenovirus E1B 19kDa interacting protein 3-like 2.226663031 A_23_P420873 NR1D1 nuclear receptor subfamily 1, group D, member 1 2.201998638 A_24_P214841 POU5F1 POU domain, class 5, transcription factor 1 2.200580047 A_23_P343104 FLJ30901 hypothetical protein FLJ30901 2.192116997 A_23_P134454 CAV1 caveolin 1, caveolae protein, 22kDa 2.179673008 A_23_P209799 MYO7B myosin VIIB 2.17953626 A_23_P154627 ZNF218 zinc finger protein 218 2.179369129 A_23_P15226 2.167793506 A_32_P175739 HK2 hexokinase 2 2.139031047 A_23_P79518 IL1B interleukin 1, beta 2.137341726 A_23_P369899 RIS1 Ras-induced senescence 1 2.093543399 A_23_P57089 TMEPAI transmembrane, prostate androgen induced RNA 2.08829922 A_23_P122104 SH3PXD2B SH3 and PX domains 2B 2.082199862 A_23_P354027 KCTD11 potassium channel tetramerisation domain containing 11 2.077675874 A_24_P319374 GPA33 glycoprotein A33 (transmembrane) 2.070062932 A_23_P360329 AIM1L absent in melanoma 1-like 2.063846817 A_23_P131846 SNAI1 snail homolog 1 (Drosophila) 2.063186082 A_24_P253078 DKFZP586D0919 hepatocellularcarcinoma-associated antigen HCA557a 2.039403328 A_32_P4608 2.036785797 A_23_P85693 GBP2 guanylate binding protein 2, interferon-inducible 2.016886953 A_23_P215790 EGFR epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) 2.013366996 A_23_P110846 CNOT8 CCR4-NOT transcription complex, subunit 8 2.010242909 A_23_P211428 SMTN smoothelin 2.007799709 A_23_P503072 CCL28 chemokine (C-C motif) ligand 28 1.992964027 A_24_P119774 PGK1 Phosphoglycerate kinase 1 1.98868748 A_32_P47643 FAM110C Family with sequence similarity 110 member C 1.985097597 A_23_P428738 ANG angiogenin, ribonuclease, RNase A family, 5 1.976742918 A_24_P410363 EGLN1 egl nine homolog 1 (C. elegans) 1.976028539 A_24_P838748 WIRE WIRE protein 1.966254894 A_23_P35796 PPP2R5B protein phosphatase 2, regulatory subunit B (B56), beta isoform 1.965129487 A_24_P323084 C17orf55 chromosome 17 open reading frame 55 1.96197779 A_23_P430051 NGLY1 N-glycanase 1 1.956631008 A_23_P24903 P2RY2 purinergic receptor P2Y, G-protein coupled, 2 1.935871503 A_24_P390096 GLIPR1 GLI pathogenesis-related 1 (glioma) 1.927995159 A_23_P48676 PYGL phosphorylase, glycogen; liver (Hers disease, glycogen storage disease type VI) 1.926251397 A_23_P121596 PPBP pro-platelet basic protein (chemokine (C-X-C motif) ligand 7) 1.923422938 A_24_P77008 PTGS2 prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) 1.922058238 A_23_P302005 STON1 stonin 1 1.913641734 A_23_P571 SLC2A1 solute carrier family 2 (facilitated glucose transporter), member 1 1.906216604 A_23_P253982 HOXA4 homeobox A4 1.904355381 A_24_P391987 LOC401152 HCV F-transactivated protein 1 1.903025234 A_24_P345290 LOC645619 Similar to Adenylate kinase isoenzyme 4, mitochondrial (ATP-AMP transphosphorylase) 1.901038228 A_32_P129340 CDNA clone IMAGE:4838568 1.893829642 A_23_P12463 QSCN6 quiescin Q6 1.881229385 A_23_P110122 CCNG2 cyclin G2 1.872986809 A_32_P171143 LOC389458 hypothetical gene supported by BC031661 1.852107179 A_23_P36825 GPRC5A G protein-coupled receptor, family C, group 5, member A 1.850772532 A_23_P110585 MGC21644 hypothetical protein MGC21644 1.849570325 A_23_P334727 NRG4 neuregulin 4 1.841854137 A_23_P78108 ALDOC aldolase C, fructose-bisphosphate 1.831732876 A_23_P250963 SLC1A3 solute carrier family 1 (glial high affinity glutamate transporter), member 3 1.830699621 A_32_P72351 FAM20C Family with sequence similarity 20, member C 1.825218452 A_32_P218806 PSIP1 PC4 and SFRS1 interacting protein 1 1.821599192 A_24_P391260 PTTG1IP pituitary tumor-transforming 1 interacting protein 1.820894452 A_32_P167239 FLJ36748 hypothetical protein FLJ36748 1.814340672 A_23_P158570 ACADSB acyl-Coenzyme A dehydrogenase, short/branched chain 1.813103342 A_23_P17065 CCL20 chemokine (C-C motif) ligand 20 1.811653671 A_23_P167096 VEGFC vascular endothelial growth factor C 1.8019005 A_23_P75310 ARHGAP22 Rho GTPase activating protein 22 1.801831796 A_23_P32404 ISG20 interferon stimulated exonuclease gene 20kDa 1.798149432 A_32_P188186 1.786579556 A_23_P208937 TLE6 transducin-like enhancer of split 6 (E(sp1) homolog, Drosophila) 1.783145343 A_24_P105761 JMJD1A jumonji domain containing 1A 1.76178275 A_23_P359015 PHF21A PHD finger protein 21A 1.756301148 A_23_P129821 KCNJ12 potassium inwardly-rectifying channel, subfamily J, member 12 1.754304671 A_23_P103110 MAFF v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian) 1.737932837 A_23_P388433 LOC401152 HCV F-transactivated protein 1 1.729456022 A_23_P130961 ELA2 elastase 2, neutrophil 1.713149563 A_24_P119567 CCDC52 coiled-coil domain containing 52 1.693109179 A_23_P30363 P4HA2 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide II 1.68336739 A_23_P68072 WDR54 WD repeat domain 54 1.681701208 A_23_P66355 ITGB4 integrin, beta 4 1.666534603 A_32_P108655 AK3L1 adenylate kinase 3-like 1 1.662623945 A_23_P39237 ZFP36 zinc finger protein 36, C3H type, homolog (mouse) 1.655158476 A_24_P20806 PRR7 proline rich 7 (synaptic) 1.640059339 A_24_P126741 1.639511377 A_23_P37856 HBA1 hemoglobin, alpha 1 1.635795681 A_23_P206707 MT1G metallothionein 1G 1.635782942 A_23_P146943 ATP1B1 ATPase, Na+/K+ transporting, beta 1 polypeptide 1.611411809 A_24_P194881 SHANK3 SH3 and multiple ankyrin repeat domains 3 1.608643439 A_23_P4662 BCL3 B-cell CLL/lymphoma 3 1.601650369 A_24_P15621 FLJ43855 similar to sodium- and chloride-dependent creatine transporter 1.589017987 A_23_P18966 P4HA2 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide II 1.58672519 A_24_P289178 MGC17624 C16orf74 1.580611302 A_23_P330070 TFPI tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) 1.563328606 A_23_P10614 PDK1 pyruvate dehydrogenase kinase, isozyme 1 1.557419736 A_23_P143845 TIPARP TCDD-inducible poly(ADP-ribose) polymerase 1.553760817 A_24_P814749 1.543179206 A_32_P55106 1.522882107 A_23_P207020 WDR45L WDR45-like 1.522694871 A_24_P944331 PAQR5 progestin and adipoQ receptor family member V 1.513073959 A_23_P137984 S100A10 S100 calcium binding protein A10 (annexin II ligand, calpactin I, light polypeptide (p11)) 1.48730829 A_23_P336554 IL1RAP interleukin 1 receptor accessory protein 1.478173861 A_23_P146077 ZNF395 zinc finger protein 395 1.45987556 A_23_P73220 FGD6 FYVE, RhoGEF and PH domain containing 6 1.454585993 A_24_P898945 C18orf19 Chromosome 18 open reading frame 19 1.421222516 A_32_P1657 cDNA DKFZp667C1617 from clone DKFZp667C1617 1.413443524 A_23_P115407 GSTM1 glutathione S-transferase M1 1.412463206

Fold change ProbeName GeneSymbol GeneName log-ratio(2%/20%) DOWN A_32_P119744 LOC344595 Hypothetical LOC344595 -3.626719362 A_23_P93141 GSTA5 glutathione S-transferase A5 -3.505034561 A_23_P30294 CDO1 cysteine dioxygenase, type I -3.217881438 A_24_P926410 PAH PAH=phenylalanine hydroxylase {exon 9/10, mutant L333F} -3.044855526 A_32_P196193 PAQR9 progestin and adipoQ receptor family member IX -2.892782322 A_23_P352266 BCL2 B-cell CLL/lymphoma 2 -2.765691012 A_32_P209230 CITED4 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 4 -2.573471342 A_24_P290087 -2.502004633 A_23_P104438 MYPN myopalladin -2.467488622 A_23_P502747 RASAL2 RAS protein activator like 2 -2.465556117 A_23_P502464 NOS2A nitric oxide synthase 2A (inducible, hepatocytes) -2.459124429 A_23_P66328 ACSM2 acyl-CoA synthetase medium-chain family member 2 -2.449742345 A_23_P69531 KLB klotho beta -2.44858084 A_24_P366555 PTHB1 parathyroid hormone-responsive B1 -2.425156977 A_24_P624439 LOC400566 hypothetical gene supported by AK128660 -2.354226016 A_23_P23996 MAT1A methionine adenosyltransferase I, alpha -2.346951595 A_23_P66698 HCP1 heme carrier protein 1 -2.344043628 A_23_P146187 RRS1 RRS1 ribosome biogenesis regulator homolog (S. cerevisiae) -2.254255494 A_32_P162726 EXOSC6 Exosome component 6 -2.216654821 A_24_P463929 LOC285733 hypothetical protein LOC285733 -2.171653532 A_24_P190007 GP2 glycoprotein 2 (zymogen granule membrane) -2.15728858 A_23_P97265 GPATC4 G patch domain containing 4 -2.156489663 A_23_P306479 clone IMAGE:4402152 -2.127523292 A_32_P83049 KIAA0953 -2.10447461 A_23_P83098 ALDH1A1 aldehyde dehydrogenase 1 family, member A1 -2.067565384 A_23_P159952 BEX1 brain expressed, X-linked 1 -2.064092934 A_23_P308032 NUP98 nucleoporin 98kDa -2.051949265 A_23_P361841 FAM80B Family with sequence similarity 80, member B -2.013916056 A_23_P4536 EPB41L3 erythrocyte membrane protein band 4.1-like 3 -1.991221481 A_32_P51237 ANKRD38 ankyrin repeat domain 38 -1.975403356 A_32_P141238 TMEM16B transmembrane protein 16B -1.953701907 A_23_P67381 SULT2A1 sulfotransferase family, cytosolic, 2A, dehydroepiandrosterone (DHEA)-preferring, member 1 -1.952868194 A_24_P290163 DKFZp547E087 hypothetical gene LOC283846 -1.888222554 A_23_P52939 SLC43A1 solute carrier family 43, member 1 -1.874751786 A_23_P151098 SPSB2 splA/ryanodine receptor domain and SOCS box containing 2 -1.869908628 A_23_P101564 FGF21 fibroblast growth factor 21 -1.845992602 A_23_P87238 SAA4 serum amyloid A4, constitutive -1.776921239 A_23_P117743 ASB7 ankyrin repeat and SOCS box-containing 7 -1.764684203 A_23_P156180 SLC22A4 solute carrier family 22 (organic cation transporter), member 4 -1.759043355 A_23_P70670 CD83 CD83 antigen (activated B lymphocytes, immunoglobulin superfamily) -1.718834287 A_32_P71518 EMILIN1 Elastin microfibril interfacer 1 -1.692982645 A_23_P79927 NOL5A nucleolar protein 5A (56kDa with KKE/D repeat) -1.690416715 A_23_P111402 RSPO3 R-spondin 3 homolog (Xenopus laevis) -1.668785589 A_23_P79591 APOB apolipoprotein B (including Ag(x) antigen) -1.666615017 A_32_P58755 Similarity_ECOLI Beta-galactosidase -1.648477558 A_23_P15876 ALPK2 alpha-kinase 2 -1.632362812 A_23_P45294 CXorf39 chromosome X open reading frame 39 -1.613931267 A_23_P9152 RCL1 RNA terminal phosphate cyclase-like 1 -1.613510963 A_23_P21706 CTPS CTP synthase -1.606098984 A_24_P66001 UCRC ubiquinol-cytochrome c reductase complex (7.2 kD) -1.594531874 A_23_P8240 FAM50B family with sequence similarity 50, member B -1.577824336 A_23_P144697 RAD1 RAD1 homolog (S. pombe) -1.572309027 A_23_P145786 MLXIPL MLX interacting protein-like -1.531612045 A_23_P501007 EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1 -1.499632619 A_23_P8119 ZBTB9 zinc finger and BTB domain containing 9 -1.483003055 A_23_P52797 TMPRSS5 transmembrane protease, serine 5 (spinesin) -1.476443667 A_23_P144684 ANKRD32 ankyrin repeat domain 32 -1.469056087 A_23_P250118 HSPBAP1 HSPB (heat shock 27kDa) associated protein 1 -1.439616904 Quantitative RT-PC and immunohistochemistry

To confirm the microarray results we performed a new set of cell culture

experiments on HepG2 cells at 20% O2 and in parallel at 2% O2. We analyzed the expression of selected genes at different time points (between 0 and 72 hours) by real-time PCR with each sample in duplicate. Real-time data at 72 hours are in agreement with microarray findings (Table 3).

2% vs 20% oxygen during 72 hours

Gene Array PCR

CCNG2 1.87 0.89

CDO1 -3.22 -1.75

EGLN1 2.01 0.93

EGLN3 3.51 1.93

ERO1L 2.59 1.06

FGF21 -1.85 -2.18

HIF1A 0.62 0.23

IGFBP3 3.71 1.99

MAT1A -2.35 -2.06

RCL1 -1.61 -0.84

VEGF 2.51 2.25

WDR45L 1.52 1.25

Table 3. Comparison of gene expression ratio (2log) from microarray and by RT-PCR for selected genes. HepG2 cells were cultured for 72 hours in

2% O2 or 20% O2, cells were collected and after RNA extraction used in microarray or RT-PCR as described in materials and method. The ratio

between expression at 2% O2 compared to expression at 20% O2 is presented in the table.

Hif1A showed a dynamic in its mRNA expression over time (Figure 1) with an induction in the first phase and adaptation after longer exposure to reduced oxygen. Most of the other genes we investigated also showed a bi-phasic response. EGLN1, IGFBP and VEGF initially went up and declined after they had peaked, CDO1 and FIH showed a gradual decrease over the whole time of the experiment. These observations support the initial assumption that the acute hypoxic state (up to 24 hrs) has a different gene expression pattern compared to the more chronic state.

Figure 1. Gene expression in cultures of HepG2 cells after exposure to hypoxia as determined by Quantitative RT-PCR. HIF1A, Hif-regulators (EGLN1 and FIH) and HIF-target gene VEGF were assayed by real time PCR. In addition we chose CDO1 and IGFBP from the list of most significantly altered genes and determined expression ratio. Expression ratio (2log) was determined in parallel cultures with β2M as housekeeping gene and expressed as increase (positive) or decrease compared to control cultures kept at 20% O2.

Figure 2. Immunohistochemical staining for Hif-1α and VEGF after exposure to normal (20%) or impaired (2%) oxygen at several time points. A) To evaluate the staining a semi-quantative quickscore (1-9) was used which combines positivity (P) with a range from 1-6 and intensity (I), with a range from 0 – 3.(4) There is a strong induction of both proteins in the acute phase (0-24 hours), but after prolonged hypoxia a new balance occurs. Hif-1α is not expressed under normal oxygen (20%) conditions, whereas VEGF has a low constitutional expression. B-D) Immunohistochemical staining under hypoxic conditions B) Hif-1α staining at 0hrs - there is no Hif-1α present. C) Hif-1α staining after 24hrs – almost all cells are positive. D) Hif-1α staining after 72hrs – some cells are positive.

7 Gene hypoxia score for HepG2 cells determined at different oxygen concentrations

CCNG2 EGLN3 ERO1L FGF21 MAT1A RCL1 WDR45L PCR 7‐gene PCR * hypoxia cut off Real‐time PCR score +/‐1

5% O2 24 hrs 0,92 3,11 1,15 nd 0,96 0,49 0,72 0,74 2/7 72 hrs ‐0,04 2,01 0,38 nd ‐1,04 ‐1,25 ‐0,31 1,66 3/7

2% O2 24 hrs 0,45 1,64 2,04 nd ‐1,13 ‐0,45 1,75 2,26 4/7 72 hrs 0,89 1,93 1,06 ‐2,18 ‐2,06 ‐0,84 1,25 2,98 5/7

1% O2 24 hrs 1,32 4,66 2,72 nd ‐0,75 ‐0,18 0,86 2,86 3/7 72 hrs 0,87 3,83 2,16 nd ‐0,61 0,04 0,59 2,15 2/7

Table 4: Gene expression ratio (2log) of HepG2 cultured under different oxygen concentrations determined by RT-PCR. Differential expression compared to parallel cultures at 20% O2. *: the number of genes that were detected when we use a cut off of 2 exp +/- 1 as was used in the microarray. nd: due to the low expression of FGF21 we could not accurately determine the differential expression of this gene under most of the conditions investigated using the commercial assay Hs00173927_m1.

Hypoxia score was determined by RT-PCR for cells cultured under different oxygen conditions. The most pronounced effect was observed at 72 hours and 2

% O2 with the highest hypoxia score and the most genes with a change in expression more than +/- 1 (5 out of 7). Cells cultured in 1% O2 for 24 hours had an almost equal hypoxia score but this was dominated by 3 genes (CCNG2,

EGLN3 and ERO1L). When HepG2 cells were cultured with 2% O2 continuously no extensive cell death was noted, the cell doubling time increased and the

cultures could be passaged by trypsinization for at least 2 months.

Liver specificity of differentially expressed genes

To determine the liver specificity of the 7-gene prognostic signature we retrieved expression data of four data sets deposited at NCBI (GDS422, GDS423,

GDS1209 and GDS1663). The data sets contain gene expression of a variety of normal tissue. A semi-quantitative expression score was made for each of the seven genes based on the values in the NCBI database. Blank= no data available, 0 = < 20% (blue), 1 = 20-50% (green) and 2 = 40-70% (orange). The mean for each gene is presented in figure 3.

Figure 3: mRNA expression of the 7 genes in normal human tissues. Blank= no data available, 0 = < 20% (blue), 1 = 20-50% (green) and 2 = 40- 70% (orange).

To gain further insight in the liver specificity of the gene set we identified by microarray we repeated the cell culture and microarray analysis using two gastrointestinal cancer cell lines: CaCo2 (ATCC, HTB-37) which is derived from colorectal adenocarcinoma(5) and HPAC (ATCC, CRL-2119) derived from pancreas carcinoma(6). Experimental procedure was similar to that used for

HepG2 and analysis was performed using the Agilent’s 44k dual color Human

Whole Genome Oligo Microarray. As criteria we used a corrected p-value below

0.05 and a fold change of >2 and in case of multiple measurements of the same probe that gene was only included once. In this way we could identify 3,259 different genes for HepG2, 3,389 genes for CaCo2 and 1,681 genes for HPAC.

Only 181 genes were differentially expressed under hypoxia in all of the three cell lines (Figure 4). Among these 181 genes there were 3 genes present (CCNG2,

EGLN3 and ERO1L) that were also found in the 7 gene set. These 3 genes do not have as high a performance to predict prognosis as the 7 gene set.

Figure 4: Overlap between the differentially expressed genes under hypoxia between liver (HepG2), colon (CaCo2) and pancreatic cancer cells (HPAC). HepG2 (ATCC, HB-8065), CaCo2 (ATCC, HTB-37) and HPAC (ATCC, CRL-2119) were cultured as described in Materials and Methods using the appropriate medium. Venn-diagram analysis was performed to show overlap in gene expression between these cell lines for the significantly altered genes (fold change >2 and p-value <0.05).

Functions and interactions of the 7 genes in relation to hypoxia

The functions of these seven genes are either related to hypoxia, to cell cycle or to metabolism.

- Cyclin G2 (CCNG2) is an unconventional cyclin expressed at modest levels in proliferating cells, peaking during the late S and early G2-phase.(7) It is significantly upregulated as cells exit the cell cycle in response to DNA damage.

cDNA microarray analyses consistently point to CCNG2 upregulation in parallel

with cell cycle inhibition during the responses to diverse growth inhibitory signals,

such as heat shock, oxidative stress and hypoxia.(8)

- EGL nine homolog 3 (EGLN3), also prolyl hydroxylase 3, is a key regulator in

chronic hypoxia. Recently it has been demonstrated that Hif-1 is not

overexpressed in chronic hypoxia due to upregulation of the different prolyl

hydroxylases. In the acute phase EGLN1 has a dominant role, whereas EGLN3

comes into play during sustained hypoxia and promotes cell survival,(9) which

supports our findings.

- Endoplasmic Reticulum Oxidoreductin-1 L (Ero1L) upregulation by hypoxia was

demonstrated before in a variety of tumor cell lines, as well as in nontransformed

primary cells, including hepatocellular carcinoma cells.(10) In the first period (6h)

this is Hif dependent, but after 12 hrs there is also a Hif-independent manner.(11)

Ero1L is necessary in the disulfide formation which is essential for the correct folding of proteins in the endoplasmic reticulum. Upregulation of Ero1L will proportionally increase the capability for proper protein folding under hypoxia in face of diminution in the ER oxidizing power due to the lack of oxygen and induces cell proliferation and survival. This response to hypoxia with upregulation of Ero1L is called the unfolded protein response (UPR) and regulates ER homeostasis and promotes hypoxia tolerance.(12)

- WDR45L which encodes for a WD-40 repeat containing protein, is a member of

a gene family involved in a variety of cellular processes, including cell cycle progression, signal transduction, apoptosis, and gene regulation. The exact

function of WDR45L is unknown, but other family members such as WDR1 and

WIPI3 are overexpressed in several human cancers.(13) WDR16 is even

overexpressed in a great majority of HCC patients and suppression leads to

growth retardation.(14)

- Fibroblast growth factor 21 (FGF21) is one of the downregulated genes in the

hypoxia signature. FGF family members possess broad mitogenic and cell

survival activities and are involved in a variety of biological processes including

cell growth, tissue repair, tumor growth and invasion. FGF 21 is a member of the

FGF_15 family. FGF21 is mainly produced in the liver and has an

endocrinological function. It has an important role in glucose, lipid and energy

homeostasis. For example, it regulates glucose uptake in adipocytes.(15)

Methionine adenosyltransferase I alpha (MAT1A) is critical for a differentiated

and functional competent liver. It serves as a key enzyme in the production of S-

adenosylmethionine, which is the source of methyl groups for most biological methylations.(16) In previous research it has been demonstrated that MAT1A is

reduced in cirrhosis and HCC.(17, 18) Underexpression of MAT1A induces cell

vulnerability to oxidative stress and facilitates the development to HCC.(19) This

gene is also underexpressed in the proliferation cluster of the two studies that

published their molecular classification for HCC.(20, 21)

- RCL1 (RNA terminal phosphate cyclase-like 1) is also underexpressed in the

proliferation cluster in both studies. The exact function of this cyclase in humans is not completely understood, but involves RNA pre-processing. In yeasts RCL1

is essential for viability and growth.(22)

To explore functional interactions or partnership between these 7 genes we

loaded them into the STRING 8 program (http://string-db.org/). This program

weights and integrates information from numerous sources, including

experimental repositories, computational prediction methods and public text collections, thus acting as a meta-database that maps all interaction evidence

onto a common set of genomes and proteins.(23) No direct link was found

between the 7 genes. When we included proven functional partners (10, in color

e.g. MOP1=HIF1A) and 15 white nodes (e.g. VEGFA) linking interactions

between the 7+10 genes, we obtained the following picture (Figure 5). There is

clear interaction between most of the seven genes in our hypoxia score and well-

known hypoxia inducible genes. MAT1A, RCL1 and WDR45L are outliers,

MAT1A is liver specific and the exact functions of RCL1 and WDR45L are

unknown.

Figure 5. Schematic representation of functional interactions obtained for the 7 gene set from STRING 8.0 computer program. The 7 prognostic hypoxia genes and were linked with predicted functional partners and 15 white nodes were included to show the most relevant interactions. A red line indicates the presence of fusion evidence; a green line - neighborhood evidence; a blue line - coocurrence evidence; a purple line - experimental evidence; a yellow line - textmining evidence; a light blue line - database evidence; a black line - coexpression evidence (for further details see http://string-db.org/).

REFERENCES TO SUPPLEMENTARY MATERIAL

1. Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome biology 2004;5: R80. 2. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology 2004;3: Article3. 3. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society: Series B 1995;57: 289-300. 4. Detre S, Saclani Jotti G, Dowsett M. A "quickscore" method for immunohistochemical semiquantitation: validation for oestrogen receptor in breast carcinomas. Journal of clinical pathology 1995;48: 876-8. 5. Trainer DL, Kline T, McCabe FL, et al. Biological characterization and oncogene expression in human colorectal carcinoma cell lines. International journal of cancer 1988;41: 287-96. 6. Gower Jr W, al. e. HPAC, a new human glucocorticoid-sensitive pancreatic ductal adenocarcinoma cell line. In Vitro Cell Dev Biol 1994;30A: 151-61. 7. Kasukabe T, Okabe-Kado J, Honma Y. Cotylenin A, a new differentiation inducer, and rapamycin cooperatively inhibit growth of cancer cells through induction of cyclin G2. Cancer science 2008;99: 1693-8. 8. Murray JI, Whitfield ML, Trinklein ND, Myers RM, Brown PO, Botstein D. Diverse and specific gene expression responses to stresses in cultured human cells. Molecular biology of the cell 2004;15: 2361-74. 9. Ginouves A, Ilc K, Macias N, Pouyssegur J, Berra E. PHDs overactivation during chronic hypoxia "desensitizes" HIFalpha and protects cells from necrosis. Proceedings of the National Academy of Sciences of the United States of America 2008;105: 4745-50. 10. May D, Itin A, Gal O, Kalinski H, Feinstein E, Keshet E. Ero1-L alpha plays a key role in a HIF-1-mediated pathway to improve disulfide bond formation and VEGF secretion under hypoxia: implication for cancer. Oncogene 2005;24: 1011-20. 11. Gess B, Hofbauer KH, Wenger RH, Lohaus C, Meyer HE, Kurtz A. The cellular oxygen tension regulates expression of the endoplasmic oxidoreductase ERO1-Lalpha. European journal of biochemistry / FEBS 2003;270: 2228-35. 12. Wouters BG, Koritzinsky M. Hypoxia signalling through mTOR and the unfolded protein response in cancer. Nat Rev Cancer 2008;8: 851-64. 13. Proikas-Cezanne T, Waddell S, Gaugel A, Frickey T, Lupas A, Nordheim A. WIPI- 1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy. Oncogene 2004;23: 9314-25. 14. Silva FP, Hamamoto R, Nakamura Y, Furukawa Y. WDRPUH, a novel WD-repeat- containing protein, is highly expressed in human hepatocellular carcinoma and involved in cell proliferation. Neoplasia (New York, NY 2005;7: 348-55. 15. Kharitonenkov A, Shiyanova TL, Koester A, et al. FGF-21 as a novel metabolic regulator. The Journal of clinical investigation 2005;115: 1627-35. 16. Mato JM, Corrales FJ, Lu SC, Avila MA. S-Adenosylmethionine: a control switch that regulates liver function. Faseb J 2002;16: 15-26. 17. Cai J, Sun WM, Hwang JJ, Stain SC, Lu SC. Changes in S-adenosylmethionine synthetase in human liver cancer: molecular characterization and significance. Hepatology (Baltimore, Md 1996;24: 1090-7. 18. Avila MA, Berasain C, Torres L, et al. Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma. Journal of hepatology 2000;33: 907-14. 19. Martinez-Chantar ML, Corrales FJ, Martinez-Cruz LA, et al. Spontaneous oxidative stress and liver tumors in mice lacking methionine adenosyltransferase 1A. Faseb J 2002;16: 1292-4. 20. Boyault S, Rickman DS, de Reynies A, et al. Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets. Hepatology (Baltimore, Md 2007;45: 42-52. 21. Chiang DY, Villanueva A, Hoshida Y, et al. Focal gains of VEGFA and molecular classification of hepatocellular carcinoma. Cancer research 2008;68: 6779-88. 22. Billy E, Wegierski T, Nasr F, Filipowicz W. Rcl1p, the yeast protein similar to the RNA 3'-phosphate cyclase, associates with U3 snoRNP and is required for 18S rRNA biogenesis. The EMBO journal 2000;19: 2115-26. 23. Jensen LJ, Kuhn M, Stark M, et al. STRING 8--a global view on proteins and their functional interactions in 630 organisms. Nucleic acids research 2009;37: D412-6.