Background
Methotrexate (MTX) is a classical drug that is used for the treatment of a wide variety of cancers, both alone and in combination with other chemotherapeutic agents
In the present report, we identified genes differentially expressed in at least 4 out of the seven cell lines. Among the genes that fulfilled this requisite, we found some genes that we had previously studied as modulators of MTX resistance, and S100A4, a gene overexpressed in five out of the seven cell lines studied.
S100A4 is a member of the S100 calcium binding protein family, which is composed of more than 20 members. Their name was given because they are soluble in 100% saturated ammonium sulfate
S100A4 has been described to be involved in a wide variety of intra- and extracellular processes, such as protein phosphorylation, dynamics of cytoskeleton components or Ca2+ homeostasis, which are regulated through interaction of S100A4 with its target proteins
In this work, we searched for genes differentially expressed in common among cell lines resistant to MTX. We identified S100A4 as a gene overexpressed in five out of the seven MTX-resistant cell lines studied, which had been previously associated with chemotherapy resistance. Functional validations using either an expression vector encoding for S100A4 or siRNA against its RNA show a role for S100A4 in MTX resistance.
Methods
Cell Lines
Cell lines representative of five types of human cancer were used: HT29 and Caco-2 of colon cancer, MCF-7 and MDA-MB-468 of breast cancer, MIA PaCa-2 of pancreatic cancer, K562 of erythroblastic leukemia, and Saos-2 of osteosarcoma
Cell Culture
Human cell lines were routinely grown in Ham's F12 medium supplemented with 7% fetal bovine serum (FBS, both from Gibco) at 37°C in a 5% CO2 humidified atmosphere. Resistant cells were routinely grown in selective DHFR medium (-GHT medium, GIBCO) lacking glycine, hypoxanthine and thymidine, the final products of DHFR activity. This medium was supplemented with 7% dialyzed fetal bovine serum (GIBCO).
Global microarray data analyses of cell lines resistant to MTX
A global comparison of all cell lines was performed using GeneSpring GX v 7.3.1 (Agilent Technologies), using the latest gene annotations available (March 2009), in order to find differentially expressed genes in the majority of the resistant cell lines. The triplicate samples for each condition, sensitive and resistant, in each of the seven cell lines (42 samples in total) were imported into one single experiment. Normalization was applied in two steps: i) "per Chip normalization" by which each measurement was divided by the 50th percentile of all measurements in its array; and ii) "per Gene normalization" by which the samples of each cell line were normalized against the median of the respective sensitive cells (control). The expression of each gene was calculated as the ratio of the value obtained for each condition relative to the control condition after normalization of the data. Then, data were filtered using the control strength, a control value calculated using the Cross-Gene Error Model on replicates
RT-Real-Time PCR
mRNA levels were determined by RT-Real-time PCR. Total RNA was extracted from cells using Ultraspec™ RNA reagent (Biotecx) following the recommendations of the manufacturer. Complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. The reaction mixture was incubated at 37°C for 60 min. The cDNA product was used for subsequent Real-time PCR amplification using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with 25 ng of the cDNA mixture and the assays-on-demand from Applied Biosystems Hs00243202_m1 for S100A4 and Hs00356991_m1 for APRT.
Gene copy number determination
Genomic DNA from either sensitive or resistant cells was obtained with the Wizard™ Genomic DNA Purification Kit (Promega) following the manufacturer's recommendations. We used 25 ng of DNA and the following primers for Real-Time PCR amplification: For S100A4: 5'-CTTCTGG-GCTGCTTAT-3' and 5'-ACTGGGCTTCTGT-TTTCTATC-3'; and for APRT: 5'-CGGGAAC-CCTCGTCTTTCGCC-3' and 5'-GCCTCGGG-GGCTCAATCTCAC-3'.
Preparation of total extracts for Western blotting
Total extracts from cells were used to assay S100A4 protein levels. Cells were scrapped in 700 μl of ice-cold PBS and centrifuged for 10 min at 2,000 rpm on a microfuge. The supernatant was discarded and cells were resupended in 50 μl of RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Igepal CA-630 (Sigma), 0.5 mM PMSF, Protease inhibitor cocktail from Sigma, 1 mM NaF; pH 7.4). Cells were incubated in ice for 30 min with vortexing every 10 minutes and then centrifuged at 15,000 × g at 4°C for 10 min. Five μl of the extract were used to determine protein concentration by the Bradford assay (Bio-Rad). The extracts were frozen in liquid N2 and stored at -80°C. Different amounts of total extracts were resolved on SDS 15%-polyacrylamide gels. After protein transference using a semidry electroblotter, PVDF membranes (Immobilon P, Millipore) were incubated with an antibody against S100A4 (DAKO), and detection was accomplished by secondary horseradish peroxidase-conjugated antibody and enhanced chemiluminescence, as recommended by the manufacturer (Amersham). To normalize the results, blots were re-probed with antibodies against either Actin or GAPDH (Sigma).
Transfection of an expression vector encoding for S100A4
Cells were seeded into 6-well plates in 1 ml of HAM's F12 selective medium. Eighteen hours later, cells were transfected with the expression plasmid for S100A4 (pCMV6-XL5-S100A4, OriGene Technologies; abbreviated in the manuscript as pCMV-S100A4), using either Lipofectamine™2000 (Invitrogen) or Fugene (Roche) following the manufacturer's specifications. The overexpression of S100A4 was monitored by determining its mRNA levels after 48 h and by determining its protein levels after 72 h upon transfection, respectively. When assaying the sensitivity to MTX caused by overexpression of S100A4, MTX was added 48 h upon transfection and cell viability determined using the MTT assay 6 days after MTX addition. An empty vector was transfected in parallel with pCMV-S100A4 and was used as negative control.
ELISA assay
Cells were transfected with pCMV-S100A4 as described above and cell culture media was collected 72 h after transfection. Sandwich ELISA was performed in MaxiSorb plates (NUNC), coated with a mouse anti-S100A4 antibody O/N at 4°C. Blocking was performed with 1% BSA for 1 h at 37°C. Incubation with the samples was performed for 2.5 h at 37°C, followed by incubation with a rabbit anti-S100A4 antibody. Finally, incubation with a secondary goat anti-rabbit horseradish peroxidase-conjugated antibody was performed for 1 h at 37°C. Reading of absorbance upon TMB addition was performed in a Multiskan Ascent (Thermo) plate-reading spectrophotometer at 630 nm. Background signal was determined in parallel and was used to correct the absorbance values. All analyses were performed at least in duplicate for each sample in each experiment.
Transfection of siRNAs against S100A4 RNA
HT29 cells were plated in 1 ml of -GHT medium. Transfection was performed eighteen hours later with a siRNA designed against S100A4 RNA (siS100A4) or with a siS100A4-4 MIS bearing 4 mismatches with respect to siS100A4 (underlined in the sequence below).
siS100A4: 5'-CAGGGACAACGAGGTGGAC-3'
siS100A4-4 MIS: 5'-CA
- CCCT
Cells were lipofected with the siRNAs using Lipofectamine™2000 (Invitrogen) in accordance to the manufacturer's instructions. MTX (10-7M) was added 48 hours after siRNA treatment and MTT assays were performed as described previously
The siRNAs were designed using the software iRNAi v2.1. Among the possible alternatives, sequences rich in A/T on the 3' of the target were chosen. Then, BLAST resources in NCBI
Primers for off-target effects determination. The word table shows the sequences of the primers used to assess the off-target effects of transfected siS100A4 by determining the mRNA levels of Enolase 2, Topoisomerase II, Clusterin and UGT1A7 by RT-Real-Time PCR. APRT mRNA was used to normalize the results.
Click here for file
Transfection of an expression vector encoding for β-catenin
Cells were seeded into 6-well plates. Eighteen hours later, transfection with the expression plasmid for β-catenin (pcDNA3-β-catenin) kindly provided by Dr. Duñach, Universitat Autònoma de Barcelona, Spain) was performed as described for pCMV-S100A4. Cells were collected 48 h after transfection for RNA determination. S100A4 mRNA levels were monitored by RT-Real-Time PCR. The empty vector was transfected in parallel with pcDNA3-β-catenin and was used as negative control.
Statistical analyses
Data are presented as mean ± SE. Statistical analyses were performed using the unpaired t test option in GraphPad InStat version 3.1a for Macintosh. p-values of less than 0.05 were considered statistically significant.
Results
S100A4 is overexpressed but not amplified in human cells resistant to methotrexate
In previous studies, we used the HG U133 PLUS 2.0 microarrays from Affymetrix as a tool to analyze the differential gene expression between sensitive and MTX-resistant cells derived from different human cell lines representative of colon cancer (HT29 and CaCo-2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2) (GEO series accession number [GSE16648], which also contains the current data). Now we performed a global analysis of the seven cell lines to find out genes differentially expressed in at least four out of the seven cell lines. The list of these genes is provided in Table
Genes differentially expressed in at least four out of the seven MTX-resistant cell lines studied.
Genbank
Gene Name
Description
Fold Change
HT29
Caco-2
MCF-7
MDA-
MB-468
MIA
PaCa-2
K562
SaOs-2
AKR1C2
aldo-keto reductase family 1, member C2
4.6
10.2
0.03
31.1
0.02
5.0
NS
AKR1C3
aldo-keto reductase family 1, member C3
1.8
4.6
0.8
73.3
0.7
12.0
NS
ALDH3A1
aldehyde dehydrogenase 3 family, member A1
2.9
3.8
6.6
11.9
0.4
NS
NS
ATG10
ATG10 autophagy related 10 homolog
8.7
NS
NS
1.5
2.1
10.1
2.0
CRIP2
cysteine-rich protein 2
2.7
1.3
1.9
NS
2.2
NS
0.3
CSRP2
cysteine and glycine-rich protein 2
2.0
0.3
2.7
NS
2.0
2.9
0.5
CYP1B1
cytochrome P450, family 1, subfamily B, polypeptide 1
3.1
2.3
0.5
NS
4.2
NS
0.4
DHFR
dihydrofolate reductase
7.3
50.2
52.8
1.8
16.9
17.8
8.9
DHFRL1
dihydrofolate reductase-like 1
7.0
10.8
5.5
NS
3.0
17.5
2.1
DKK1
dickkopf homolog 1 (Xenopus laevis)
4.3
2.5
2.1
NS
0.7
NS
19.1
ELL2
elongation factor, RNA polymerase II, 2
NS
1.7
0.4
2.6
8.0
2.3
2.7
EPAS1
endothelial PAS domain protein 1
2.8
NS
2.4
NS
6.4
0.4
0.1
EPB41L4A
erythrocyte membrane protein band 4.1 like 4A
1.6
1.3
1.7
1.4
2.3
4.4
4.7
FAM46A
family with sequence similarity 46, member A
NS
2.6
NS
NS
3.9
2.5
1.5
FGFR3
fibroblast growth factor receptor 3
NS
4.6
3.1
0.4
1.5
0.4
3.4
FLJ10357
hypothetical protein FLJ10357
NS
3.8
NS
NS
2.1
3.2
3.5
HOMER1
homer homolog 1 (Drosophila)
NS
NS
27.9
NS
2.4
13.8
NS
KLK5
kallikrein-related peptidase 5
2.3
NS
2.2
NS
3.5
NS
4.6
LOC92270
V-type proton ATPase subunit S1-like protein
54.0
2.3
NS
NS
3.4
25.0
NS
MLLT11
myeloid/lymphoid or mixed-lineage leukemia
2.2
2.6
NS
NS
1.8
0.08
1.7
MSH3
mutS homolog 3 (E. coli)
6.3
8.2
3,8
NS
13.2
35.9
1,5
PECI
peroxisomal D3, D2-enoyl-CoA isomerase
7.5
2.6
1.9
NS
NS
NS
1.8
PSMD11
proteasome 26 S subunit, non-ATPase, 11
NS
2.3
NS
NS
1.3
3.5
1.4
RGS2
regulator of G-protein signaling 2, 24 kDa
1.6
8.2
3.6
NS
3.2
1.9
NS
RPS23
ribosomal protein S23
12.9
NS
NS
NS
2.9
8.9
1.8
S100A2
S100 calcium binding protein A2
0.5
0.3
7.1
NS
4.0
NS
4.1
S100A4
S100 calcium binding protein A4
3.7
0.6
4.3
NS
2.4
1.5
2.5
SLC2A13
solute carrier family 2, member 13
4.0
9.4
0.6
2.5
4.3
10.2
NS
SLC6A6
solute carrier family 6, member 6
0.6
2.6
2.2
NS
2.4
1.5
1.5
SMARCA1
SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 1
2.6
2.6
14.0
NS
NS
3.4
1.7
SSBP2
single-stranded DNA binding protein 2
10.6
NS
0.5
NS
2.9
5.0
2.0
STXBP6
syntaxin binding protein 6 (amisyn)
NS
2.1
27.5
NS
3.0
14.5
0.5
THBS4
thrombospondin 4
NS
12.9
9.2
NS
15.7
4.9
0.6
UGT1A6
UDP glucuronosyltransferase 1, polypeptide A6
NS
2.8
24.4
27.9
5.3
NS
NS
VIM
vimentin
2.4
NS
3.6
NS
NS
4.9
1.7
ZCCHC9
zinc finger, CCHC domain containing 9
8.3
1.5
0.5
NS
6.7
7.9
NS
ZFYVE16
zinc finger, FYVE domain containing 16
6.1
45.8
76.4
NS
16.5
23.1
3.2
A global comparison analysis was performed to find out differentially expressed genes in the majority of the MTX-resistant cells. The table includes the list of the genes differentially expressed in at least four out of the seven MTX-resistant cell lines that displayed a Benjamini-Hochberg false discovery rate-corrected p-value < 0.05. For each gene, the Genbank accession number, gene name, description and fold change in each resistant cell line with respect to its respective sensitive counterpart are shown. All values displayed are significant, unless otherwise stated (NS, non significant; p-value > 0.05)
Validation of S100A4 overexpression and copy number determination in the different cell lines.
Cell Line
Expression
Microarray
Signal Sensitive
Signal Resistant
Ratio
RT-PCR Validation
Copy- number
Protein
HT29
1876
6862
3.7
6.2 ± 0.2
1.4 ± 0.1
22.5 ± 1.0
MCF-7
49
213
4.3
6.2 ± 0.3
1.5 ± 0.1
10.8 ± 2.2
MiaPaCa-2
3304
7925
2.4
4.7 ± 0.6
1.2 ± 0.1
24.4 ± 8.1
K562
147
249
1.5
3.6 ± 0.2
1.6 ± 0.1
4.0 ± 1.0
Saos-2
137
345
2.5
2.2 ± 0.1
2.16 ± 0.07
2 ± 0.4
CaCo-2
4515
2769
0.6
N/D
N/D
N/D
MDA-MB-468
317
235
0.7
N/D
N/D
N/D
The table shows the S100A4 expression levels obtained in the microarray experiments for all the cell lines studied. The level of expression is presented as the mean of the signal value in sensitive and resistant cells, as well as the ratio between them (fold change). Gene overexpression was validated in five of the seven cell lines by RT-Real-Time PCR.
Ectopic overexpression of S100A4 desensitizes HT29 cells toward MTX
HT29 MTX-resistant cells displayed the highest S100A4 expression values, considering both the mRNA and protein levels (Table
Effects on S100A4 expression and MTX sensitivity upon pCMV-S100A4 transfection of HT29 cells
Effects on S100A4 expression and MTX sensitivity upon pCMV-S100A4 transfection of HT29 cells. A) mRNA levels of S100A4 determined by RT-Real-Time PCR 48 h after treatment of HT29 cells (30,000) with 250 ng of the expression vector for S100A4 (pCMV-S100A4). B) A representative image of the intracellular protein levels of S100A4 determined by Western Blotting 72 h after ectopic transfection with its expression vector is shown in the upper panel, and the quantification of the blots is shown in the lower panel. Purified S100A4 protein was used as a reference marker (Abnova; first lane). An additional panel showing endogenous S100A4 protein levels in HT29 sensitive (S) and resistant (R) cells is also provided. C) Effects of S100A4 overexpression on cell viability. HT29 cells (100,000) were treated with 1 μg of pCMV-S100A4 and 5 × 10-8M MTX was added 48 h later. Cell viability was assessed by the MTT assay six days after MTX treatment. D) Extracellular S100A4 protein levels quantified by ELISA 72 h after S100A4 overexpression upon pCMV-S100A4 transfection. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p < 0.05, **p < 0.01, *** p < 0.001.
S100A4 is secreted by HT29 cells transfected with pCMV-S100A4
Given that it had been described that S100A4 has extracellular functions
Knocking down S100A4 with siRNA chemosensitizes HT29 toward MTX
We used iRNA technology to study the role of S100A4 in MTX resistance. Treatment of sensitive HT29 cells with increasing concentrations (10-100 nM) of the siRNA against S100A4 (siS100A4) showed a progressive decrease in its mRNA levels (Figure
Assessment of siS100A4 specificity.
Gene
S100A4
Enolase 2
Topoisomerase II
Clusterin
UGT1A7
Control
100
100
100
100
100
siRNA S100A4
18.6 ± 6
135.8 ± 21.6
127.5 ± 6.4
133.9 ± 14.4
159.5 ± 28.4
HT29 cells were transfected with 100 nM siS100A4. Forty-eight hours later, the mRNA levels of S100A4 and four unrelated genes were determined by RT-Real-Time PCR. Data represent the mean of three experiments ± SE.
Effects on S100A4 expression and MTX sensitivity upon siS100A4 transfection of HT29 cells
Effects on S100A4 expression and MTX sensitivity upon siS100A4 transfection of HT29 cells. A) HT29 cells (30,000) were transfected with siS100A4 as described in Methods. Total RNA was extracted after 48 h and S100A4 mRNA levels were determined by RT-Real-Time PCR. B) S100A4 protein levels were determined by Western Blotting 72 h after transfection, using specific antibodies against S100A4 and Actin to normalize the results. C) Chemosensitization assays toward methotrexate: cells were treated with siS100A4 for 48 h and then incubated with MTX. Cell viability was determined 3 days after MTX treatment. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. A representative image of Western Blots is presented. *p < 0.05, ** p < 0.01, *** p < 0.001.
Treatment with 100 nM siS100A4 was also performed in HT29 MTX-resistant cells. This approach led to a reduction of 75% in the levels of S100A4 RNA (Figure
Effects on S100A4 expression and MTX sensitivity upon siS100A4 transfection of HT29 MTX-resistant cells
Effects on S100A4 expression and MTX sensitivity upon siS100A4 transfection of HT29 MTX-resistant cells. A) S100A4 mRNA levels were determined by RT-Real-time PCR as described in Methods 48 h after siS100A4 treatment. B) Cells were treated with MTX after a 48 h-pre-incubation with siS100A4, and cell viability was determined 3 days later by the MTT assay. The levels of expression and the viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p < 0.05, ** p < 0.01.
S100A4 is transcriptionally regulated by the Wnt pathway in HT29 cells
It had been previously described that S100A4 was a target of the Wnt signaling pathway and a functional TCF binding site has been identified in its promoter sequence
Effects of transfecting an expression vector encoding for β-Catenin on S100A4 mRNA levels
Effects of transfecting an expression vector encoding for β-Catenin on S100A4 mRNA levels. Transfection with β-Catenin expression vector (pcDNA3-β-Catenin) was performed in HT29 cells, both sensitive (Figure 4A) and resistant (Figure 4B) as described in Methods. S100A4 mRNA levels were determined by RT-Real-Time PCR 48 h after transfection. All results are expressed as percentages referred to untreated cells. Values are the mean of three independent experiments ± SE. * p < 0.05.
Discussion
The principal aim of this work was to find out genes differentially expressed in cell lines resistant to MTX representative of five different types of human cancer. In a previous report
The human S100A4 is a member of the S100 family of EF-hand Ca2+-binding proteins. The multigenic family of S100 proteins is expressed in vertebrates exclusively and has been described to play intracellular and extracellular regulatory activities on protein phosphorylation, on the dynamics of cytoskeleton components or on Ca2+ homeostasis
We tried to mimic the overexpression of S100A4 in the resistant cells by transfecting HT29 sensitive cells with an expression vector encoding for S100A4. We tested that S100A4 mRNA and intracellular protein levels were significantly increased after transfection. Importantly, we could observe a reversion of the cytotoxicity caused by MTX upon transfection with S100A4 expression vector. It is worth mentioning that a moderate S100A4 overexpression has been described in a colon cancer cell line resistant to doxorubicin
To investigate the role of intracellular S100A4 on MTX resistance, we used iRNA technology. Transfection of a siRNA against S100A4 in HT29 MTX-resistant cells caused a reduction in gene RNA levels but did not alter cell viability. The latter effect could be explained by the overexpression by gene amplification of the
In an attempt to determine the mechanism for the overexpression of S100A4 in HT29 resistant cells, we determined S100A4 copy number in both HT29 cell lines. Although it has been previously described that the chromosomal region where the gene is located (1q21) is amplified in breast cancer
Although many biological functions have been attributed to S100A4, the exact molecular mechanisms by which S100A4 exerts these functions have not been fully elucidated
S100A4 has been reported to interact with p53, thus interfering in p53 activation and DNA binding capacity
In summary, our results show a role for S100A4 in MTX resistance. Its overexpression in HT29 MTX-resistant cells is not due to changes in gene copy-number but to a transcriptional regulation, probably through the Wnt pathway. Cellular knockdown of S100A4 leads to chemosensitization toward MTX and ectopic gene overexpression desensitizes the cells toward this chemotherapeutic agent.
Conclusions
S100A4 was identified and confirmed as a gene overexpressed in five out of the seven MTX-resistant cell lines studied. S100A4 ectopic overexpression in HT29 cells lead to a desensitization toward MTX. Conversely, transfection experiments using interference RNA produced a chemosensitization to MTX. The results obtained in this report establish a relation between S100A4 and resistance to MTX.
Abbreviations
MTX: methotrexate; DHFR: dihydrofolate reductase; S100A4: S100 calcium binding protein A4.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NM, ES, CA, XV, IR, JA, SR, and JLH performed the experimental work. ES and CJC analyzed the data. VN and CJC designed the study, helped with data interpretation and supervised the experimental work. All authors wrote and approved the final manuscript.