SUPPLEMENTARY DATA

SUPPLEMENTARY FIGURES:

Supplementary Figure S1: Correlations of the 2-week change in the breast cancer hypoxia metagene with the 2-week change in AvERG and TFF1/pS2

A. Two-week change in the breast cancer hypoxia metagene versus 2-week change in

AvERG. B. Two-week change in the breast cancer hypoxia metagene versus 2-week change in TFF1/pS2.

1 SUPPLEMENTARY TABLES:

Supplementary Table S1: Annotation of the proliferation metagene

Gene Accession symbol Number name apolipoprotein B mRNA editing , catalytic

APOBEC3B NM_004900 polypeptide-like 3B asp (abnormal spindle) homolog, microcephaly associated ASPM NM_018136 (Drosophila) AURKB NM_004217 aurora kinase B BIRC5 NM_001012270 baculoviral IAP repeat-containing 5 BUB1 NM_004336 budding uninhibited by benzimidazoles 1 homolog (yeast) budding uninhibited by benzimidazoles 1 homolog beta BUB1B NM_001211 (yeast) C21orf45 NM_018944 21 open 45 CCNA2 NM_001237 cyclin A2 CCNB1 NM_031966 cyclin B1 CCNB2 NM_004701 cyclin B2 CCNE2 NM_057749 cyclin E2

CDC2 NM_001170407 cyclin-dependent kinase 1

CDC23 NM_004661 cell division cycle 23, yeast, homolog CDC7 NM_001134419 cell division cycle 7 homolog (S. cerevisiae)

CDCA3 NM_031299 cell division cycle associated 3

CDCA8 NM_018101 cell division cycle associated 8 CDKN3 NM_001130851 cyclin-dependent kinase inhibitor 3 CENPA NM_001042426 centromere A CENPF NM_016343 centromere protein F, 350/400kDa (mitosin) CEP55 NM_001127182 centrosomal protein 55kDa CKAP2 NM_001098525 cytoskeleton associated protein 2 CKS2 NM_001827 CDC28 protein kinase regulatory subunit 2 CLCC1 NM_001048210 chloride channel CLIC-like 1 COP9 constitutive photomorphogenic homolog subunit 6 COPS6 NM_006833 (Arabidopsis) CSE1L NM_001316 CSE1 chromosome segregation 1-like (yeast) CTPS NM_001905 CTP synthase DDX39 NM_005804 DEAD (Asp-Glu-Ala-Asp) box polypeptide 39

DLG7 NM_014750 discs, large (Drosophila) homolog-associated protein 5 DNAJC9 NM_015190 DnaJ (Hsp40) homolog, subfamily C, member 9 DONSON NM_017613 downstream neighbor of SON DTL NM_016448 denticleless homolog (Drosophila) ECT2 NM_018098 epithelial cell transforming sequence 2 oncogene elongation of very long chain fatty acids (FEN1/Elo2, ELOVL1 NM_022821 SUR4/Elo3, yeast)-like 1 ENOSF1 NM_001126123 enolase superfamily member 1

2 EZH2 NM_004456 enhancer of zeste homolog 2 (Drosophila) FBXO5 NM_001142522 F-box protein 5 FEN1 NM_004111 flap structure-specific 1 FOXM1 NM_021953 forkhead box M1 gamma-glutamyl (conjugase, GGH NM_003878 folylpolygammaglutamyl hydrolase) GMNN NM_015895 geminin, DNA replication inhibitor HMGB2 NM_001130688 high-mobility group box 2 HMGB3 NM_005342 high-mobility group box 3 HMMR NM_001142556 hyaluronan-mediated motility receptor (RHAMM) HSPA14 NM_016299 heat shock 70kDa protein 14 KIAA0101 NM_001029989 KIAA0101 KIF11 NM_004523 kinesin family member 11 KIF14 NM_014875 kinesin family member 14 KIF23 NM_004856 kinesin family member 23 KIF2C NM_006845 kinesin family member 2C KIF4A NM_012310 kinesin family member 4A KNTC1 NM_014708 kinetochore associated 1 MAD2L1 NM_002358 MAD2 mitotic arrest deficient-like 1 (yeast) MCM2 NM_004526 minichromosome maintenance complex component 2 MCM6 NM_005915 minichromosome maintenance complex component 6 MCM7 NM_005916 minichromosome maintenance complex component 7 MELK NM_014791 maternal embryonic leucine zipper kinase MKI67 NM_001145966 antigen identified by monoclonal antibody Ki-67 MPP1 NM_001166460 membrane protein, palmitoylated 1, 55kDa MRPL47 NM_020409 mitochondrial L47

MRS2L NM_020662 MRS2 magnesium homeostasis factor homolog-like MSH2 NM_000251 mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) MYBL2 NM_002466 v-myb myeloblastosis viral oncogene homolog (avian)-like 2 NEK2 NM_002497 NIMA (never in mitosis gene a)-related kinase 2 NUP155 NM_004298 nucleoporin 155kDa NUSAP1 NM_001129897 nucleolar and spindle associated protein 1 OIP5 NM_007280 Opa interacting protein 5

ORC6L NM_014321 origin recognition complex, subunit 6 PBK NM_018492 PDZ binding kinase POLE2 NM_001197330 (DNA directed), epsilon 2 (p59 subunit) PRC1 NM_003981 protein regulator of cytokinesis 1 PSME4 NM_014614 proteasome (prosome, macropain) activator subunit 4

RACGAP1 NM_001126103 Rac GTPase activating protein 1 RAD51AP1 NM_001130862 RAD51 associated protein 1 RFC3 NM_002915 replication factor C (activator 1) 3, 38kDa RFC4 NM_002916 replication factor C (activator 1) 4, 37kDa RPL39L NM_052969 ribosomal protein L39-like RPP40 NM_006638 P/MRP 40kDa subunit RRM2 NM_001034 ribonucleotide reductase M2

3 SEPHS1 NM_001195602 selenophosphate synthetase 1 solute carrier family 4, sodium bicarbonate cotransporter,

SLC4A5 NM_021196 member 5 SRPK1 NM_003137 SRSF protein kinase 1 STIL NM_001048166 SCL/TAL1 interrupting locus STMN1 NM_001145454 stathmin 1 TACC3 NM_006342 transforming, acidic coiled-coil containing protein 3 TFRC NM_001128148 transferrin receptor (p90, CD71) TOP2A NM_001067 (DNA) II alpha 170kDa TPX2 NM_012112 TPX2, microtubule-associated, homolog (Xenopus laevis) TRIP13 NM_001166260 thyroid hormone receptor interactor 13 TTK NM_001166691 TTK protein kinase UBE2C NM_007019 ubiquitin-conjugating enzyme E2C UNG NM_003362 uracil-DNA glycosylase VRK1 NM_003384 vaccinia related kinase 1 WDR12 NM_018256 WD repeat domain 12 ZWINT NM_001005413 ZW10 interactor

Supplementary Table S2: Proliferation-associated in the original breast cancer metagene

Gene Accession Gene name symbol number ADM NM_001124 Adrenomedullin BTG3 NM_006806 BTG family, member 3 BUB1 NM_004336 BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) CCNB1 NM_031966 Cyclin B1 CCNB2 NM_004701 Cyclin B2 CDC2 NM_033379 Cell division cycle 2, G1 to S and G2 to M CDC20 NM_001255 Cell division cycle 20 homolog (S. cerevisiae) CDKN3 NM_005192 Cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity ) CEP55 NM_018131 Centrosomal protein 55kDa DLG7 NM_014750 Disc large (Drosophila) homolog-associated protein 5 GGH NM_003878 Gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) HMGB3 NM_005342 High-mobility group box 3 KIF4A NM_012310 Kinesin family member 4A MAD2L1 NM_002358 MAD2 mitotic arrest deficient-like 1 (yeast) MCTS1 NM_014060 Malignant T cell amplified sequence 1 MELK NM_014791 Maternal embryonic leucine zipper kinase MIF NM_002415 Macrophage migration inhibitory factor (glycosylation- inhibiting factor) NUP155 NM_004298 Nucleoporin 155kDa

4 PTTG1 NM_004219 Pituitary tumor-transforming 1 RACGAP1 NM_013277 Rac GTPase activating protein 1 RRM2 NM_001034 Ribonucleotide reductase M2 polypeptide STK6 NM_198434 Aurora kinase A TFRC NM_003234 Transferrin receptor (p90, CD71) TPX2 NM_012112 TPX2, microtubule-associated, homolog (Xenopus laevis)

Supplementary Table S3: Annotation of the breast cancer hypoxia metagene excluding proliferation-associated genes

Gene symbol Accession Gene name Meta- number connectivity score PGAM1 NM_002629 Phosphoglycerate mutase 1 (brain) 1.00 GARS NM_002047 Glycyl-tRNA synthetase 1.00 BNIP3 NM_004052 BCL2/adenovirus E1B 19kDa interacting 1.00 protein 3 LDHA NM_005566 Lactate dehydrogenase A 1.00 P4HA1 NM_000917 Procollagen-proline, 2-oxoglutarate 4- 1.00 dioxygenase (proline 4-hydroxylase), alpha polypeptide I GPI NM_000175 Glucose phosphate 0.99 NDRG1 NM_006096 N-myc downstream regulated 1 0.99 GAPDH NM_002046 glyceraldehyde-3-phosphate 0.99 dehydrogenase DDIT4 NM_019058 DNA-damage-inducible transcript 4 0.99 VEGF NM_001025366 vascular endothelial growth factor A 0.99 PFKP NM_002627 Phosphofructokinase, platelet 0.99 LOC388642 XM_934933 TPI1 pseudogene 0.99 PGK1 NM_000291 Phosphoglycerate kinase 1 0.99 ENO1 NM_001428 Enolase 1, (alpha) 0.98 DSCR2 NM_203433 proteosome assembly chaperone 1 0.98 SLC16A3 NM_004207 Solute carrier family 16, member 3 0.98 (monocarboxylic acid transporter 4) PRDX4 NM_006406 Peroxiredoxin 4 0.98 SLC2A1 NM_006516 Solute carrier family 2 (facilitated glucose 0.98 transporter), member 1 AK3L1 NM_016282 Adenylate kinase 3 like 1 0.97 GOLT1B NM_016072 Golgi transport 1 homolog B (S. 0.97 cerevisiae) RANBP1 NM_002882 RAN binding protein 1 0.97 RALA NM_005402 V-ral simian leukemia viral oncogene 0.97 homolog A (ras related) RIS1 NM_015444 transmembrane protein 158 0.97 SEC61G NM_001012456 Sec61 gamma subunit 0.97 ENY2 NM_020189 Enhancer of yellow 2 homolog 0.97

5 (Drosophila) MRPS17 NM_015969 Mitochondrial ribosomal protein S17 0.97 MTFR1 NM_014637 Mitochondrial fission regulator 1 0.97 MRPL15 NM_014175 Mitochondrial ribosomal protein L15 0.97 ANKRD37 NM_181726 Ankyrin repeat domain 37 0.97 CTSL2 NM_001333 Cathepsin L2 0.97 SLC7A5 NM_003486 Solute carrier family 7 (cationic amino 0.97 acid transporter, y+ system), member 5 MMP1 NM_002421 Matrix metallopeptidase 1 (interstitial 0.96 collagenase) PSMB5 NM_002797 Proteasome (prosome, macropain) 0.96 subunit, beta type, 5 TMEM70 NM_017866 Transmembrane protein 70 0.96 IMPAD1 NM_017813 Inositol monophosphatase domain 0.96 containing 1 PDIA6 NM_005742 Protein disulfide isomerase family A, 0.96 member 6 MRPL13 NM_014078 Mitochondrial ribosomal protein L13 0.96 IL8 NM_000584 interleukin 8 0.96 MTCH2 NM_014342 Mitochondrial carrier homolog 2 (C. 0.96 elegans) PSMA5 NM_002790 Proteasome (prosome, macropain) 0.95 subunit, alpha type, 5 KIF20A NM_005733 Kinesin family member 20A 0.95 ATP1B3 NM_001679 ATPase, Na+/K+ transporting, beta 3 0.95 polypeptide ATP5G3 NM_001002256 ATP synthase, H+ transporting, 0.95 mitochondrial F0 complex, subunit C3 (subunit 9) UBE2S NM_014501 Ubiquitin-conjugating enzyme E2S 0.95 COX4NB NM_006067 COX4 neighbor 0.95 RBM35A NM_017697 RNA binding motif protein 35A 0.95 EIF4EBP1 NM_004095 Eukaryotic 4E 0.95 binding protein 1 TCEB1 NM_005648 Transcription B (SIII), 0.95 polypeptide 1 (15kDa, elongin C) NP NM_000270 Nucleoside phosphorylase 0.95 CHCHD2 NM_016139 coiled-coil-helix-coiled-coil-helix domain 0.95 containing 2 SF3B5 NM_031287 Splicing factor 3b, subunit 5, 10kDa 0.95 NUP93 NM_014669 Nucleoporin 93kDa 0.95 RNASEH2A NM_006397 Ribonuclease H2, subunit A 0.95 C6orf129 NM_138493 open reading frame 129 0.95 LSM4 NM_012321 LSM4 homolog, U6 small nuclear RNA 0.94 associated (S. cerevisiae) IMPA2 NM_014214 Inositol(myo)-1(or 4)-monophosphatase 2 0.94 MTHFD2 NM_006636 Methylenetetrahydrofolate dehydrogenase 0.94 (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase

6 EIF2S2 NM_003908 initiation factor 2, 0.94 subunit 2 beta, 38kDa NFIL3 NM_005384 Nuclear factor, interleukin 3 regulated 0.94 GMPS NM_003875 Guanine monphosphate synthetase 0.94 SRD5A1 NM_001047 Steroid-5-alpha-reductase, alpha 0.94 polypeptide 1 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1) PSMD8 NM_002812 Proteasome (prosome, macropain) 26S 0.94 subunit, non-ATPase, 8 YEATS2 NM_018023 YEATS domain containing 2 0.94 C16orf61 NM_020188 Chromosome 16 open reading frame 61 0.94 KIF18A NM_031217 Kinesin family member 18A 0.94 MGC40489 XM_940001 Similar to pleckstrin homology domain 0.93 containing, family M (with RUN domain) member 1; adapter protein 162 LRRC59 NM_018509 Leucine rich repeat containing 59 0.93 HCCS NM_005333 Holocytochrome c synthase (cytochrome 0.93 c heme-) PLOD1 NM_000302 Procollagen-lysine 1, 2-oxoglutarate 5- 0.93 dioxygenase 1 SHMT2 NM_005412 Serine hydroxymethyltransferase 2 0.93 (mitochondrial)

Supplementary Table S4: Genes in the breast hypoxia metagene and their expression changes in cell lines experiments of hypoxia exposure and

HIF1a/HIF2a siRNAs [1]. Genes are ranked by their meta-connectvity scores

(from highest to lowest).

EXPERIMENT 1: EXPERIMENT 2: Hypoxia or DMOG versus silencing of HIF1a and Normoxia HIF2a in hypoxia

Gene symbol Accession P.value P.value number PGAM1 NM_002629 <10-4 <10-4 GARS NM_002047 0.7768 0.1407 BNIP3 NM_004052 0.0001 0.0036 LDHA NM_001135239 0.0001 0.0005 P4HA1 NM_000917 <10-4 <10-4 GPI NM_000175 0.0004 0.0015 NDRG1 NM_001135242 <10-4 <10-4 GAPDH NM_002046 0.0034 0.5930 DDIT4 NM_019058 0.0002 0.0546 VEGF NM_001025366 <10-4 0.0328 PFKP NM_002627 <10-4 <10-4

7 LOC388642 XM_934933 0.0065 0.0173 PGK1 NM_000291 <10-4 0.0248 ENO1 NM_001428 0.0003 <10-4 DSCR2 NM_203433 0.0842 <10-4 SLC16A3 NM_001042422 0.0078 0.5559 PRDX4 NM_006406 0.4557 0.5581 SLC2A1 NM_006516 0.0057 0.0002 AK3L1 NM_203464 NA 0.0055 GOLT1B NM_016072 0.6781 0.1747 RANBP1 NM_002882 0.0029 0.1352 RALA NM_005402 0.7244 0.6847 RIS1 NM_015444 0.4234 0.0199 SEC61G NM_001012456 0.0094 0.0001 ENY2 NM_001193557 0.4024 0.9658 MRPS17 NM_015969 0.1253 0.0188 MTFR1 NM_001145838 0.1095 0.0026 MRPL15 NM_014175 0.1158 0.0003 ANKRD37 NM_181726 NA 0.1172 CTSL2 NM_001333 0.0637 0.6626 SLC7A5 NM_003486 0.0938 0.1914 MMP1 NM_001145938 0.5695 0.7762 PSMB5 NM_001130725 0.0295 0.1939 TMEM70 NM_001040613 0.0283 0.0036 IMPAD1 NM_017813 0.9519 0.0045 PDIA6 NM_005742 0.9748 0.0026 MRPL13 NM_014078 0.9231 0.4343 IL8 NM_000584 0.4151 0.8363 MTCH2 NM_014342 0.1602 0.3426 PSMA5 NM_002790 0.0126 0.4974 KIF20A NM_005733 0.7704 0.4631 ATP1B3 NM_001679 0.0315 0.0002 ATP5G3 NM_001002258 0.1780 0.0208 UBE2S NM_014501 0.0398 0.0185 COX4NB NM_001142288 0.0001 <10-4 RBM35A NM_001034915 0.3424 0.2926 EIF4EBP1 NM_004095 0.0157 0.5265 TCEB1 NM_005648 0.4733 0.2400 NP NM_000270 0.1420 0.0002 CHCHD2 NM_016139 0.7556 0.9752 SF3B5 NM_031287 0.1179 0.3086 NUP93 NM_014669 0.0089 <10-4 RNASEH2A NM_006397 0.0986 0.4827 C6orf129 NM_138493 NA 0.4636 LSM4 NM_012321 0.3326 0.6605 IMPA2 NM_014214 0.3098 0.1854 MTHFD2 NM_006636 0.5843 0.0020

8 EIF2S2 NM_003908 0.1576 0.0318 NFIL3 NM_005384 0.0003 0.0122 GMPS NM_003875 0.1759 0.8230 SRD5A1 NM_001047 0.7888 0.0380 PSMD8 NM_002812 0.0051 0.0067 YEATS2 NM_018023 0.0002 <10-4 C16ORF61 NM_020188 0.0313 0.0044 KIF18A NM_031217 0.3905 0.4080 MGC40489 XM_940001 0.0472 0.0713 LRRC59 NM_018509 0.0035 0.0009 HCCS NM_001122608 0.0092 0.0402 PLOD1 NM_000302 0.0065 <10-4 SHMT2 NM_001166356 0.4049 0.2337

Supplementary Table S5: Literature search on the genes in the breast hypoxia metagene (not significantly regulated by hypoxia using a published in-vitro experiment [21]). Genes are ranked by their meta-connectivity scores (from highest to lowest)

Gene symbol Association with hypoxia from published literature The expression of GARS changed significantly as a result of low oxygen GARS treatment [2]. ANKRD37 ANKRD37 was validated experimentally as a novel HIF-1 target [3]. CTSL2 contains potential HIF binding sites as predicted by MatInspector 8.01 CTSL2 (Genomatix) [4]. The expression of SLC7A5 was up-regulated by hypoxia in monocyte- SLC7A5 derived macrophages in vitro [5]. The expression of MMP1 was markedly enhanced in fibroblast-like MMP1 synoviocytes that were cultured under hypoxia [6]. MRPL13 contains potential HIF binding sites as predicted by MatInspector 8.01 MRPL13 (Genomatix) [4]. The expression of IL8 was found to be induced by hypoxia in human endothelial cells [7], and it was markedly enhanced in fibroblast-like IL8 synoviocytes that were cultured under hypoxia [6]. KIF20A contains potential HIF binding sites as predicted by MatInspector 8.01 KIF20A (Genomatix) [4]. RBM35A was found to be significantly regulated by hypoxia in renal proximal RBM35A tubule epithelial cells [8]. TCEB1 was found to bind to von Hippel-Lindau tumor suppressor protein (pVHL) and to posttranscriptionally regulate the accumulation of hypoxia- TCEB1 inducible mRNAs under normoxic (21% O2) conditions [9]. CHCHD2 contains potential HIF binding sites as predicted by MatInspector CHCHD2 8.01 (Genomatix) [4]. IMPA2 The expression of IMPA2 was found to be changed after 6days of hypoxia [10].

9 GMPS was associated with hypoxia in microvascular coronary endothelial cells GMPS [11].

Supplementary Table 6a: Correlations of the pre-treatment levels of the combined hypoxia metagene (head and neck with breast cancers) as well as the breast cancer specific hypoxia metagene

Pre-treatment Pre-treatment 2-week Ki67 2-week Ki67 (log2) proliferation (log2) percentage metagene change in Ki67

Pre-treatment r = 0.38 r = 0.30 r = 0.34 rs = 0.21 combined hypoxia P =7x10-4 P = 0.007 P = 0.004 P = 0.08 metagene (breast cancers with head and neck cancers)

Pre-treatment r = 0.55 r = 0.67 r = 0.43 rs = 0.22 breast cancer P < 10-4 P < 10-4 P = 2x10-4 P = 0.06 hypoxia metagene

Supplementary Table 6b: Pearson correlations for the post-treatment levels of the combined hypoxia metagene (head and neck with breast cancers) as well as the breast cancer specific hypoxia metagene

2-week Ki67 (log2) 2-week post-treatment proliferation metagene

2-week post-treatment r = 0.27 r = 0.39 combined hypoxia P = 0.02 P = 3x10-4 metagene (breast cancers with head and neck cancers)

2-week post-treatment r = 0.58 r = 0.71 breast cancer hypoxia P < 10-4 P < 10-4 metagene

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