Supplementary Table 1: Microarray datasets. Fourteen human and one mouse CRC microarray data sets analyzed in this study from the Gene Expression Omnibus (GEO) and the ArrayExpress databases. The GEO or ArrayExpress database identifier (ID), species, microarray platform, number of individual samples run on the array (sample#), associated publication (Author), Institute of origin and the PubMed identifier for the associated publication (PMID) are provided.
Gene UPL Probe # Gene Description Primer (F&R) PRRX1 18 PHOX1, PMX1, PRX1 paired related homeobox 1
AGTCACCGGGACTGACCA TCTTCTCCTCAGAGTTCAACTGG
FAP 81 DKFZp686G13158, DPPIV, FAPA fibroblast activation protein, alpha
GCTACAAAGCCAAGTACTATGCAC CATGGAGGGTGGAAATGG
INHBA 62 EDF, FRP inhibin, beta A GGGAGTGATCCCTGGAAAC TCCTCTTCATGGTATTGGCACT
SULF1 66 FLJ30905, FLJ38022, FLJ41750, HSULF-1, KIAA1077, SULF-1 sulfatase 1
AAGAGTCACCTTCACCCCTTC GCTGAAGTTTGCTATCCACCTC
ANTXR1 76 ATR, FLJ10601, FLJ11298, FLJ21776, TEM8 anthrax toxin receptor 1
TGACCGAGTGTCTGTGATGAG CTTGGCTGGCTGACTGTTCT
ASPN 82 FLJ20129, OS3, PLAP-1, PLAP1, SLRR1C asporin
ACAGGGTGGATAAATTCTACTTTGA GCCTGTGTACAAGTCCAACCT
VCAN 76 CSPG2, DKFZp686K06110, ERVR, GHAP, PG-M, WGN, WGN1 versican
TGGGATTGAAGACACTCAGGA TGGCTGCCCTGTAGTGAAA
HOPX 68 CAMEO, HOD, HOP, LAGY, MGC20820, NECC1, OB1, SMAP31, TOTO HOP homeobox
ACCACGCTGTGCCTCATC GCGCTGCTTAAACCATTTCT
GERM1 63 CKTSF1B1, DAND2, DRM, gremlin, IHG-2, MGC126660 gremlin 1
ACCCACGGAAGTGACAGAAT CCCTCAGCTGTTGGCAGTAG
WISP1 51 CCN4, WISP1c, WISP1i, WISP1tc WNT1 inducible signaling pathway protein 1
CTGACTTCCAGGCATGAGG ATTGTTGTGGGGGTTGGAG
TWIST1 58 ACS3, bHLHa38, BPES2, BPES3, CRS1, H-twist, SCS, TWIST twist homolog 1 (Drosophila)
AGCTACGCCTTCTCCGTCT TCCTTCTCTGGAAACAATGACA
COL12A1 80 BA209D8, COL12A1L, DJ234P15 collagen, type XII, alpha 1 CCAAGTGGGAAACCAGGA CCTTTTAAGCCAGATTGACCA
ANGPTL2 1 ARP2, HARP, MGC8889 angiopoietin-like 2 CACCGTGAGGCATGTGAA GGTTGGTCTTTATGAACTGAAAGC
PTRF 51 CAVIN, cavin-1, CGL4, FLJ90031 polymerase I and transcript release factor
CGCAACTTCAAAGTCATGATCTAC CAGGTGAAAGGGCTCAGTCT
RCN3 52 RLP49 reticulocalbin 3, EF-hand calcium binding domain
GCTACGTGCAGGTGGAAGAG ACTGCTGCCGCTCTGTCT
BNC2 83 BSN2, DKFZp686A01127, FLJ20043, FLJ34928 basonuclin 2
CTTAAAAGAGATGCACGTCTGC TGCAGGTTTATATTGGCACTGT
MRC2 1 CD280, CLEC13E, ENDO180, FLJ35911, KIAA0709, UPARAP mannose receptor, C type 2
CTTGCTTCTCGGGGGACT GGCTGAAGATGAGGAAGACATC
COL3A1 49 EDS4A, FLJ34534 collagen, type III, alpha 1 TCCCCTGGAATCTGTGAATC TGAGTCGAATTGGGGAGAAT
MGP 2 MGLAP, NTI matrix Gla protein
TCACGAAAGCATGGAGTCCT TGAGGGGACATAAAGGTGTTG
THBS3 32 MGC119564, MGC119565, TSP3 thrombospondin 3 AGGAGACGACTCCATTCACAA GTGAGCTGGGTGACCAATG
MXRA8 18 Matrix-remodelling associated 8 GCTGAGTAAGACAAAGCACGTC GGTCCCTGAGGACAGGACT
Pmm1 33 Pmm1-001 Phosphomannomutase 1 GGGTGGCTCTGACTACTCTAAGAT ACACGTAGTCAAACTTCTCAATGACT
NFATc1 50 MGC138448, NF-ATC, NFAT2, NFATc nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1
TCCAAAGTCATTTTCGTGGA CTTTGCTTCCATCTCCCAGA
NFATc2
5
NF-ATP, NFAT1, NFATp nuclear factor of activated T-cells, cytoplasmic, calcineurin-Dependent 2
CAACGGAAAGAGGAAACGAA CTGTCTTGATGGCAGGGACT
NFATc3 49 NFAT4, NFATX nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3
GGGGCAGTGAAAGCCTCT GCTTTTCACTATAGCCCAGGAG
NFATc4 52 NF-ATc4, NFAT3 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4
CATTGAGAGGGGTCCTGATG CTCTGCAGTCGGTTCACTGT
Supplementary Table 2. Probes and primer details for the mouse putative NFAT target genes analyzed in MC38Met cells.
1
Gene Assay ID UPL Probe # Gene Description Primer (F&R) PRRX1 120630 18 PHOX1, PMX1, PRX1 paired related homeobox 1
GCACAGGCGGATGAGAAC TTCTTCTGAGTTCAGCTGGTCA
FAP 108274 19 DKFZp686G13158, DPPIV, FAPA fibroblast activation protein, alpha
TGGCGATGAACAATATCCTAGA ATCCGAACAACGGGATTCTT
INHBA 103779 12 EDF, FRP inhibin, beta A CGGGTATGTGGAGATAGAGGAT CGTCTTCCTGGCTGTTCCT
SULF1 104935 69 FLJ30905, FLJ38022, FLJ41750, HSULF-1, KIAA1077, SULF-1 sulfatase 1
AGACCTAAGAATCTTGATGTTGGAA CCATCCCATAACTGTCCTCTG
ANTXR1 119405 76 ATR, FLJ10601, FLJ11298, FLJ21776, TEM8 anthrax toxin receptor 1
TGGGTCCTACTGAGGAAAGG GACCCTGGTGAAGTTGATGC
ASPN 102994 119 FLJ20129, OS3, PLAP-1, PLAP1, SLRR1C asporin TTGCAGAAGCAAAACTGACC AAAATCCTCAAGTTCCACTGTTG
VCAN 114454 148 CSPG2, DKFZp686K06110, ERVR, GHAP, PG-M, WGN, WGN1 versican
CTTCCCTCCCCCTGATAGC CGATGGTTGTAGCCTCTTTAGG
HOPX 117171 63 CAMEO, HOD, HOP, LAGY, MGC20820, NECC1, OB1, SMAP31, TOTO HOP homeobox
ACCACGCTGTGCCTCATC AGGCGCTGCTTAAACCATT
GERM1 103109 66 CKTSF1B1, DAND2, DRM, gremlin, IHG-2, MGC126660 gremlin 1
CCGCCGCACTGACAGTAT GCACCTTGGGACCCTTTC
WISP1 110191 106 CCN4, WISP1c, WISP1i, WISP1tc WNT1 inducible signaling pathway protein 1
GATGTGGACATCCATACACTCATT GATAGGAGCGTGTGCTGATG
TWIST1 110770 58 ACS3, bHLHa38, BPES2, BPES3, CRS1, H-twist, SCS, TWIST twist homolog 1 (Drosophila)
AGCTACGCCTTCTCGGTCT TCCTTCTCTGGAAACAATGACA
COL12A1 103045 55 BA209D8, COL12A1L, DJ234P15 collagen, type XII, alpha 1
TGGCTTCAGCATTCCCTTAG AGGTTTCGAGGTGCTCCTTT
ANGPTL2
129765 75 ARP2, HARP, MGC8889 angiopoietin-like 2 GCCCACTATGCCCACTCTC CTGCAGGCAGTCTCTCCAT
PTRF 129834 1 CAVIN, cavin-1, CGL4, FLJ90031 polymerase I and transcript release factor
CAGCGTCAACGTGAAGACC CCGGCAGCTTCACTTCAT
RCN3 133901 65 RLP49 reticulocalbin 3, EF-hand calcium binding domain
GGGACATCGTGATTGCTGA CTCCTCCACCTGGACATAGC
BNC2 131147 34 BSN2, DKFZp686A01127, FLJ20043, FLJ34928 basonuclin 2
TGTTGTGGGGTTGATACATCTC TGTCTCTCTTTCTCTCACATCCA
MRC2 127182 57 CD280, CLEC13E, ENDO180, FLJ35911, KIAA0709, UPARAP mannose receptor, C type 2
AACTTCCGGGACAGTCTGG TTACAGGGACTGTCGTTCCA
COL3A1 103052 131 EDS4A, FLJ34534 collagen, type III, alpha 1 ACTGGAGCACGGGGTCTT TCCTGGTTTCCCACTTTCAC
MGP 115210 2 MGLAP, NTI matrix Gla protein
TGAAAGCATGGAATCTTATGAACTT CTGCTGAGGGGATATGAAGG
THBS3 112464 112 MGC119564, MGC119565, TSP3 thrombospondin 3 TGAGCAATCCTACCCAGACA TTGTCCTTGGTGTCCTGATG
B2M 102065 117 beta-2-microglobulin CCGTGTGAACCATGTGACTTT CCTCCATGATGCTGCTTACA
MXRA8 - 5 Matrix-remodelling associated 8 CCAGAAGTCGGGAAAGTCAA CTCACTCCTGTAAAGCATCTGG
PGK1 102083 69 MGC117307, MGC142128, MGC8947, MIG10, PGKA phosphoglycerate kinase 1
GGAGAACCTCCGCTTTCAT GCTGGCTCGGCTTTAACC
GAPDH 101128 60 G3PD, GAPD, MGC88685 glyceraldehyde-3-phosphate dehydrogenase
CTCTGCTCCTCCTGTTCGAC ACGACCAAATCCGTTGACTC
NFATc1 111948 69 MGC138448, NF-ATC, NFAT2, NFATc nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1
GGTCTTCGGGAGAGGAGAAA TGACGTTGGAGGATGCATAG
NFATc2 135948 44 Nuclear factor of activated T-cells" cytoplasmic " calcineurin-dependent 2
CATCTAACCCCATCGAGTGC GCTGTCTGTGTCTTGTCTTTCAA
NFATc3 110896 128 "Nuclear factor of activated T-cells" cytoplasmic " calcineurin-dependent 3
TCTACTGGGGGACATCCTGT CATTTGTAGATTTATTGGCTTTTCG
NFATc4 111033 52 Nuclear factor of activated T-cells" cytoplasmic " calcineurin-dependent 4
CATTGAGAGGGGTCCTGATG CTCTGCAGTCGGTTCACTGT
Supplementary Table 3. Probes and primer details for the RT-Ready plates used for putative NFAT target genes analyzed in human CRC patients RNA.
Thermoscientific Identification Target sequence
siRNA J-054700-05, NFATC1 GAAUCGAGAUCACCUCCUA
siRNA J-054700-06, NFATC1 CCACAGGCCUCGUAUCAGU
siRNA J-054700-07, NFATC1 UAUACCAGCUCUGCCAUUG
siRNA J-054700-08, NFATC1 ACGGUUACUUGGAGAAUGA
Supplementary Table 4. ON-TARGETplus SMARTpool siRNA L-054700-00-0005-05 targeting Mouse NFATc1. ON-TARGETplus Non-Targeting pool (Cat# D-001810-10-20)
Gene Symbol Full name Functional category
ANGPTL2*# Angiopoeitin-like protein Matrix associated protein
ANTXR1 Anthrax toxin receptor Actin reorganization,
ASPN*# Asporin Bone mineralization, morphogen
BNC2* Basonuclin Nuclear factor
COL12A1# Collagen 12A1 Matrix protein
COL3A1*# Collagen 3A1 Matrix protein
FAP*# Fibroblast Activation Protein Matrix protein
GREM1# Gremlin Morphogen
HOPX* HOP homeobox Transcription factor
INHBA Inhibin A Morphogen
MGP*# Matrix Gla Protein Matrix protein
MRC2*# Mannose receptor Endocytosis
MXRA8# Matrix remodeling associated protein Matrix remodeling
PRRX1* Paired Wingless homeobox Transcription factor
PTRF*# Polymerase 1 and transcript release factor Transcription factor
RCN3* Reticulocalbin ER protein, Ca2+ binding
SULF1# Sulfatase I Matrix remodeling enzyme
THBS3* Thrombospondin Matrix associated protein
TWIST1*# Twist Morphogenesis
VCAN# Versican Matrix protein
WISP1# Wnt-inducible signaling protein Cell adhesion/growth
Supplementary Table 5: Twenty-one putative NFAT transcriptional target genes. Gene symbols are paired with the full name of each gene and an assigned functional category is given for each. # Genes significantly down with siNFATc1 treatment, * significantly down with FK506 treatment, *# significantly down with both siNFATc1 and FK506 treatment.
Gene MC38Par-1 MC38Par-2 MC38Par-3 MC38Met-1 MC38Met-2 MC38Met-3
logfold change
Fold change
moderated-t statistic p value FDR
NFATC1 6.8559 7.0236 6.4217 8.2539 8.3239 8.4554 1.5773 2.9842 10.7830 2.40E-06 0.0001
NFATC2 3.3507 3.4645 3.1843 3.3224 2.8869 3.7502 -0.0133 -1.0093 -0.0698 0.9459 0.9652
NFATC3 4.0747 4.4118 3.9329 4.2507 4.2948 4.0186 0.0482 1.0340 0.3628 0.7253 0.8141
NFATC4 5.9712 5.6826 6.135 6.6945 6.2729 6.4402 0.5396 1.4536 3.8193 0.0043 0.0176
Supplementary Table 6: Summary statistics of microarray-based differential mRNA expression of NFAT family of transcription factors in three biological replicates of MC38Par and MC38Met cell lines. Statistical significance for differential expression only for NFATc1 (bolded and highlighted in red) and NFATc4 (bolded) was achieved in this analysis.
8-gene signature 16-gene signature (Ref 15)
29-gene signature (Ref 25)
149-gene signature (Ref 26)
52-gene signature (Ref 27)
449-gene signature (Ref 24)
MGPCOL3A1PTRFANGPTL2FAPMGPPTRFTWIST1
MGP ASPN COL3A1 COL3A1
PTRFANGPTL2
FAP
ASPNCOL3A1
ANGPTL2 FAPMGPPTRFTWIST1
Supplementary Table 7: Overlapping genes between 8-gene signature and previously published colon cancer specific prognostic signatures. Overlapping genes in referenced published gene signatures are highlighted alongside the NFAT-directed 8-gene signature described for metastasis in this model.
Cell Line Mice median LW/BW x100 p value (Mann Whitney) Median Flux (photon/sec) Fold Flux p value (Mann Whitney) Incidence Fisher's Test
MC38Par 6 6.58 p=0.014 2.45E+09 1.00 p=0.13 5/6 ns
MC38Met 7 10.95 8.27E+09 3.37 7/7
MC38Par+VEC 14 5.84 p=0.0004 2.97E+08 1.00 p<0.0001 8/14 p=0.017
MC38Par+NFATc1 12 20.74 1.29E+10 43.48 12/12
MC38Met+shSCR 15 6.28 p=0.0003 3.35E+07 9.39 p=0.03 11/15 p=0.026
MC38Met+shNFATc1 14 4.57 3572000 1.00 14-Apr
Supplementary Table 8. Summary statistics for in vivo work using isogenic MC38 cell lines in C57BL6/J mice.
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