SUPPLEMENTAL DATA

NFAT3 Transcription Factor inhibits Breast Cancer Cell Motility by targeting the LCN2 gene.

Marjorie Fougère, Benoît Gaudineau, Jérome Barbier, Frédéric Guaddachi, Jean-Paul Feugeas, Didier Auboeuf and Sébastien Jauliac.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Antibodies, cell lines and reagents

The following antibodies were used: anti-T7 (Novagen), anti-NFAT3 (Santa Cruz; ABR), anti-actin (Santa Cruz), anti-NFAT1 (ABR), anti-NFAT2 (ABR), anti-NFAT4 (Santa Cruz), anti-NFAT5 (ABR), anti-ER (Santa Cruz), anti-LCN2 (Abnova), anti-GST (UBI). 17-estradiol (E2) was from Sigma-Aldrich and recombinant human LCN2 from R&D (reference: 1757-LC-050). The human Lipocalin-2/NGAL Quantikine ELISA Kit was from R&D (reference: DLCN20).

All cell lines were from the ATCC. The MDA-MB-231, MDA-MB-435, MDA-MB-453 and NIH3T3 lines were maintained in Dulbecco modified Eagle medium (Biological Industries), while the T-47D, ZR-75-1 and BT-474 lines were cultured in RPMI 1640 (Biological Industries). Media were supplemented with 10% fetal calf serum (Biological Industries) except for the NIH3T3 line for which new-born calf serum (Biological Industries) was used instead. All media were supplemented with 2 mM L-glutamine (Biological Industries), penicillin (100 U/mL) and streptomycin (100μg/mL) (Invitrogen). For some experiments, cells were culture with stripped serum (Hyclone) in media without red phenol (Invitrogen).

The appropriate plasmids were transiently transfected using Exgen 500 (Euromedex) according to the manufacturer’s instructions.

Plasmids

The constitutive nuclear form of NFAT3 (NFAT3) was constructed by deleting 342 N-terminal amino acids containing the serines required for its cytosolic retention (Molkentin et al., 1998). The full-length, active form and mutant NFAT3 were generated by PCR using the human NFAT3 coding sequence and cloned in pcDNA3 vector expressing the Tag T7 in N-terminal. The LCN2 promoter was generated by PCR from genomic DNA using the following primers containing a KpnI site and cloned in pGL3 luciferase reporter vector (Promega, WI) using the following primer:

sense: 5’- GGTACCCTCGAGGGCAGGGATCCAGTGGGTGCC-3’

antisense: 5’- GGTACCGGTGGCCCTATTTATGGGATCTAGGG-3’

All constructions were verified by sequencing. ERA- and ERA-A/B- expressing and control vectors and ERE Luciferase vector have been previously described (Lazennec et al., 2001), ERA-A/B is a deleted form of ERA lacking the N-terminal domain A/B necessary for the hormone-independent action of ERA (Dutertre and Smith, 2003; Fowler et al., 2004). The pCS2-(n)--gal has been already described (Jauliac et al., 2002). The plasmid expressing the dominant negative form of Rac (Rac-N17) tagged with the GST epitope was previously described (Shaw et al., 1997).

Primers sequences used for NFAT RNA isotypes detection

For NFAT1:

5’-GACTGCATCTAACCCCATCGAG-3’ (sense, exon 5),

5’-CAGATCAGAGTGGGGTCATATTC-3’ (antisense, exon 9), product size: 402 bp

For NFAT2:

5’-GTCCCCTACGTCCTACATGAG-3’ (sense, exon 2),

5’- CGGAGTTTCTGAGTTTCAGGATTC-3’) (antisense, exon5), product size: 423 bp

For NFAT3:

5’-AGTGAACCGACTGCAGAGCAAC-3’ (sense, exon 7),

5’- AGGTCTCGGCCAATGATCTCACTC-3’ (antisense, exon 10), products sizes: upper product 760 bp, lower product 440 (alternative spliced product)

For NFAT4:

5’-CAAACCAAAGCCTGGCCACACC-3’ (sense, exon 2),

5’-CTTCCCTTCTACCTCCCACTGAG-3’ (antisense, exon 7), product size: 742 bp

For NFAT5:

5’-CGGACTTCATCTCATTGCTCAG-3’ (sense, exon 1),

5’ CCACCGCTTGTCTGACTCATTG-3’ (antisense, exon 3), products sizes: 262 bp (Dalski et al., 2000), 208 bp (exon 2 spliced), 174 bp (new splice variant, under characterisation)

For GAPDH:

5’-CTCAGACACCATGGGGAAGGTGA-3’ (sense)

5’-ATGATCTTGAGGCTGTTGTCATA-3’ (antisense), product size: 450 bp

Primers sequences used for quantification by Real-Time PCR

For human LCN2:

5’-GGTAGGCCTGGCAGGGAATG-3’ (sense)

5’- CTTAATGTTGCCCAGCGTGAAC-3’ (antisense)

For Human housekeeping gene B2M:

5’-TGCTGTCTCCATGTTTGATGTATCT-3’ (sense)

5’-TCTCTGCTCCCCACCTCTAAGT-3’ (antisense).

For human FASN:

5’-CAGGCACACACGATGGAC-3’ (sense)

5’-CGGAGTGAATCTGGGTTGAT-3’ (antisense)

For human LPL:

5’-ATGTGGCCCGGTTTATCA-3’ (sense)

5’-CTGTATCCCAAGAGATGGACATT-3’ (antisense)

For human Adiponectin:

5’-GGTGAGAAGGGTGAGAAAGGA-3’ (sense)

5’-TTTCACCGATGTCTCCCTTAG-3’ (antisense)

For human Leptin

5’-TTGTCACCAGGATCAATGACA-3’ (sense)

5’-GTCCAAACCGGTGACTTTCT-3’ (antisense)

siRNA sequence used

All siRNA were from Dharmacon

For human NFAT3, the siRNA were:

siRNA1 5’-GCGACGAGGUGGAGUCUGAUU-3’ (sense)

siRNA1 5’-AAUCAGACUCCACCUCGUCGU-3’ (antisense)

siRNA2 5’-UUGCGAAACUCCUUACCUAUU-3’ (sense)

siRNA2 5’-AAUAGGUAAGGAGUUUCGCAA-3’ (antisense)

For NFAT3, siRNA control was from Dharmacon and is a validated non targeting control

5’- UAGCGACUAAACACAUCAAUU -3’ (sense)

5’- AAUUGAUGUGUUUAGUCGCUA-3’ (antisense)

For human LCN2, the siRNA were a smartpool from Dharmacon

siRNA1 5’-UGGGCAACAUUAAGAGUUAUU-3’ (sense)

siRNA1 5’-UAACUCUUAAUGUUGCCCAUU-3’ (antisense)

siRNA2 5’-GAGCUGACUUCGGAACUAAUU-3’ (sense)

siRNA2 5’-UUAGUUCCGAAGUCAGCUCUU-3’ (antisense)

siRNA3 5’-GAAGACAAAGACCCGCAAAUU-3’ (sense)

siRNA3 5’-UUUGCGGGUCUUUGUCUUCUU-3’ (antisense)

siRNA4 5’-GAAGACAAGAGCUACAAUGUU-3’ (sense)

siRNA4 5’-CAUUGUAGCUCUUGUCUUCUU-3’ (antisense).

For LCN2, siRNA control was a validated non targeting smartpool from Dharmacon. Sequence was not provided by the manufacturer (reference Dharmacon: D-001810-02-20)

Statistical analysis.

Statistical analyses were performed using the Student’s t test for paired data. P< 0.05 was considered significant.

SUPPLEMENTAL FIGURES

Figure S1.

NFAT4 and NFAT5 but not NFAT1 are expressed in T-47D cells.Total cell lysates from MDA-MB-231 (ERA-) and T-47D (ERA+) cells were immunoblotted with specific antibodies anti-NFAT1, anti-NFAT4, anti-NFAT5 or antibody anti-actin as control (bottom).

Figure S2.

Western blot for NFAT3, NFAT2, NFAT1 and actin of total lysates of T-47D cells (ERA+) 48 hours post-transfection with siRNA directed against endogenous NFAT3 (15nM). As positive control for NFAT2 total lysate of Jurkat cells were included and for NFAT1 total lysate of MDA-MB-231.

Figure S3.

Ectopic expression of ΔNFAT3 blocks the increased invasion induced by depletion of endogenous NFAT3. T-47D cells were transiently transfected with siRNA directed against endogenous NFAT3 (siRNA NFAT3), control siRNA and/or ΔNFAT3 or the vector. The ability of these transfected cells to migrate was tested as described in Figure 1, c. Aliquots of total transfected-cell lysates were lysed and immunoblotted with an anti-NFAT3 antibody or a control anti-actin antibody. bars, SE.∗, P < 0.05.

Figure S4.

Comparaison of the levels of endogenous NFAT3 to ectopic NFAT3. T-47D (ERA+) cells were transiently transfected with NFAT3 tagged with the T7 epitope. 24 hours after transfection whole cell lysates of the transfected cells and MDA-MB-231 (negative control) were separated on SDS-PAGE and immunoblotted with an antibody directed against the endogenous NFAT3 or a control anti-actin antibody. The NFAT3 antibody is able to recognize both the endogenous and the transfected NFAT3. We show two exposition times: at 15 seconds, only the over-expressed NFAT3 was detectable and at 15 minutes endogenous NFAT3 was detectable in T-47D cells transfected with the empty vector.

Figure S5.

T-47D (ERA+) cells were transfected with NFAT3 or NFAT3 or vector control (vector). 24 hours after transfection, cytosolic (C) and nuclear extracts (N) were obtained with the NE-PER kit as directed by the manufacturer and separated on SDS-PAGE and immunoblot with an anti-T7.

Figure S6.

Representation of the LCN2 promoter used in our study. 6 potential NFAT binding sites have been found using the software TESS available on the net: http://www.cbil.upenn.edu/cgi-bin/tess/tess.

Figure S7.

MDA-MB-231 (ERA-) and T-47D (ERA+) were co-transfected with the pCS2-(n)--gal plasmid and a ERE reporter plasmid previously described (Nawaz et al., 1999)  co-transfected w either control vector or NFAT3 or ERA alone or in combination using Exgen 500. 48 h later, cells were analyzed for Luciferase and -gal activities using the Luciferase assay and Galacton-plus system, and measured in a luminometer. Quantification of ERE reporter luciferase activity normalized against that of -galactosidase activity is shown. Immunoblottings with anti-T7 (NFAT3) or anti-ERA is shown. bars, SE. , P < 0.05.

Figure S8.

T-47D and MDA-MB-231 cells were transfected with siRNA control or siLCN2 for 48 hr and apoptosis was assayed by annexin V labeling. Error bars indicate +/- SD. (left panel). Whole cell lysates of T-47D transfected with siRNA control or siLCN2 for 48 hr were separated on SDS-PAGE and immunoblot with an anti-ERA and an anti-actin.

Figure S9.

T-47D (ERA+) or MDA-MB-231 (ERA-) were treated with 15 M recombinant LCN2 (LCN2) or its vehicle (PBS) for 48 hours. After the 48 hours of treatment total RNA was isolated and Q-PCR was performed for the following LCN2 target genes: FASN, LPL, Adiponectin and Leptin. All results are representative of three independent experiments. bars, SE. , P < 0.05. , P < 0.01.

Figure S10.

T-47D (ERA+) cells were transiently transfected with Rac1 (Rac-N17) tagged with the GST epitope or control vectors (vector) and tested for their ability to migrate in presence of 15M recombinant LCN2 (LCN2) or vehicle (PBS). Immunoblotting with anti-GST and anti-actin is shown. All results are representative of three independent experiments. bars, SE. , P < 0.05.

Figure S11.

Proposed working model for NFAT3, LCN2 and ERA action on motility. In ERA+, NFAT3 prevents, through an unknown factor X, LCN2 mRNA transcription. ERA targets migratory genes that cooperate with down-regulation of LCN2 to inhibit migration. Inhibition of LCN2 is not sufficient to block the invasion in ERA+ and ERA- cells, therefore NFAT3 inhibits key invasive genes to reduce the invasive capacity.

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