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.