Oncogene (2014), 1–12 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE

A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4 MT Hansen1,8, B Forst1,8, N Cremers2,3, L Quagliata2, N Ambartsumian1,4, B Grum-Schwensen1, J Klingelho¨fer1,4, A Abdul-Al1, P Herrmann5, M Osterland5, U Stein5, GH Nielsen6, PE Scherer7, E Lukanidin1, JP Sleeman2,3,9 and M Grigorian1,4,9 S100A4 is implicated in metastasis and chronic inflammation, but its function remains uncertain. Here we establish an S100A4dependent link between inflammation and metastatic tumor progression. We found that the acute-phase response proteins serum amyloid A (SAA) 1 and SAA3 are transcriptional targets of S100A4 via Toll-like receptor 4 (TLR4)/nuclear factor-kB signaling. SAA proteins stimulated the transcription of RANTES (regulated upon activation normal T-cell expressed and presumably secreted), G-CSF (granulocyte-colony-stimulating factor) and MMP2 (matrix metalloproteinase 2), MMP3, MMP9 and MMP13. We have also shown for the first time that SAA stimulate their own transcription as well as that of proinflammatory S100A8 and S100A9 proteins. Moreover, they strongly enhanced tumor cell adhesion to fibronectin, and stimulated migration and invasion of human and mouse tumor cells. Intravenously injected S100A4 protein induced expression of SAA proteins and cytokines in an organ-specific manner. In a breast cancer animal model, ectopic expression of SAA1 or SAA3 in tumor cells potently promoted widespread metastasis formation accompanied by a massive infiltration of immune cells. Furthermore, coordinate expression of S100A4 and SAA in tumor samples from colorectal carcinoma patients significantly correlated with reduced overall survival. These data show that SAA proteins are effectors for the metastasis-promoting functions of S100A4, and serve as a link between inflammation and tumor progression. Oncogene advance online publication, 27 January 2014; doi:10.1038/onc.2013.568 Keywords: inflammation; metastasis; S100A4; serum amyloid A

INTRODUCTION The microenvironment surrounding tumor cells, including cells of the immune system and proinflammatory factors, have a key role in regulating metastasis formation.1,2 Although different subclasses of immune cells can inhibit or promote metastasis, chronic inflammation in tumors generally predicts poor prognosis.3 Serum amyloid A (SAA) is a family of highly homologous acute-phase proteins whose expression and accumulation in the blood is observed during inflammation and has been associated with tumor progression and reduced survival in many human cancers.4 SAA1 and SAA2 are the major acutephase plasma isoforms. A third acute-phase isoform SAA3 is expressed in rodents, but is a transcribed pseudogene in humans.5 In vivo concentrations of SAAs increase in the blood during the response to trauma, infection, inflammation and neoplasia, and serve to regulate homeostatically lipid metabolism and transport, immune cell chemotaxis and other inflammatory processes. SAA proteins regulate the expression of cytokines including interleukins, granulocyte-colony-stimulating factor (G-CSF) and tumor necrosis factor-a, as well as matrix metalloproteinases

(MMPs).6–12 SAA can signal via Toll-like receptors (TLRs) and nuclear factor-kB (NF-kB).9,13 In mice, SAA3 contributes to the establishment of lung premetastatic niches,14 which are microenvironments that foster metastasis formation.15 We have previously implicated the S100 family member S100A4 in both tumor progression and in inflammatory diseases such as rheumatoid arthritis, psoriasis and dermato/polymyosites.16–18 S100 proteins require calcium-induced oligomerization for their activity, and are involved in the regulation of proliferation, survival, differentiation and motility.19,20 S100A4 expression is enhanced in highly metastatic CSML100 mouse mammary adenocarcinoma cells compared with poorly metastatic CSML0 cells, and functionally contributes to metastasis.21–25 Furthermore, the metastatic behavior of the mouse adenocarcinoma cell line VMR is determined by the stroma, and is stimulated by S100A4expressing fibroblasts, indicating that S100A4 can promote metastasis formation through both autocrine and paracrine mechanisms.26 Clinical studies also demonstrate that augmented expression of S100A4 in primary tumors correlates with poor prognosis (reviewed in Helfman et al.27). S100A4-mediated

1 Danish Cancer Society Research Center, Copenhagen, Denmark; 2Universita¨tsmedizin Mannheim, University of Heidelberg, Mannheim, Germany; 3KIT Karlsruhe, EggensteinLeopoldshafen, Germany; 4Neuro-Oncology Group, Laboratory of Neural Plasticity, Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, Copenhagen University, Copenhagen, Denmark; 5Experimental and Clinical Research Center, Charite´ Universita¨tsmedizin Berlin, at the Max-Delbru¨ck Center for Molecular Medicine, Berlin, Germany; 6Air Liquide Danmark A/S, Horsens, Denmark and 7Touchstone Diabetes Center, University of Texas Southwestern Medical Center, Dallas, TX, USA. Correspondence: Dr M Grigorian, Neuro-Oncology Group, Laboratory of Neural Plasticity, Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, Copenhagen University, Blegdamsvej 3B, Copenhagen 2200, Denmark. E-mail: [email protected] 8 These authors contributed equally to this work. 9 These authors contributed equally to this work. Received 3 July 2013; revised 6 December 2013; accepted 7 December 2013

S100A4-induced SAA expression promotes metastasis MT Hansen et al

2 metastasis is associated with extensive T-cell infiltration into both the primary tumor and sites of metastasis.28 Despite the extensive evidence implicating S100A4 in metastasis formation, the mechanism by which it exerts its effects remains uncertain. Here we found that SAA proteins are transcriptional targets of S100A4. In turn, SAA proteins stimulated both their own transcription and that of a variety of cytokines and MMPs, and promoted chemotactic migration and adhesion to fibronectin by tumor cells. Ectopic SAA expression in experimental tumors sufficed to augment strongly metastasis formation, and was accompanied by a marked immune cell infiltration. Consistently, coexpression of SAA and S100A4 correlated with poor prognosis in human colorectal cancer patients. These data provide an important link between the metastasis-promoting and proinflammatory effects of S100A4, and suggest that the induction of SAA expression is a major means by which S100A4 exerts these effects. RESULTS S100A4 induces expression of proinflammatory genes, including SAA family members To investigate how S100A4 promotes metastasis, we used microarrays to transcriptionally profile VMR tumor cells treated or non-treated for 24 h with the active multimeric form of the recombinant human S100A4 (hS100A4) protein. Of the 75 genes upregulated in response to hS100A4, more than 30% are associated with inflammation (Table 1). The most strongly upregulated genes included the acute-phase reactants (SAA1 and SAA3), cytokines and their receptors (CXCL1, CSF1, CCL4, INFAR2), the proinflammatory S100 family member S100A8, inflammationassociated proteases and immune cell-specific genes. Given their upregulation in response to S100A4 and the literature correlating their expression with poor prognosis, we determined whether SAA1 and SAA3 (hereafter referred to generically as SAA) might be mediators of S100A4-induced metastasis. First, we validated the transcriptional profiling. In VMR cells, SAA1 and SAA3 were both strongly transcriptionally induced in response to hS100A4 protein, as assessed by quantitative real-time polymerase chain reaction (qPCR) (Figures 1a and b). SAA1/2 and SAA3 proteins were also detected in conditioned medium (CM) from cells 24 h after induction (Figures 1a and b). Next, we examined whether S100A4-mediated upregulation of SAA proteins also occurs in human tumor cells. As human SAA3 is a pseudogene, we only monitored the expression of SAA1. Treatment of a panel of human cancer cells with hS100A4 protein also resulted in the accumulation of SAA1/2 in the CM (Figure 1c). In primary mouse bone marrow-derived macrophages, we found strong transactivation of SAA3 but not SAA1 by S100A4 (Supplementary Figure S1A). In T cells, basal expression of both SAAs was very weak, and their transactivation in response to S100A4 was negligible (Supplementary Figure S1B). Polymixin B sulfate (PMB), a potent endotoxin inhibitor, had no significant effect on the hS100A4-driven upregulation of SAA3 (Supplementary Figure S1C) but completely neutralized the even stronger induction of SAA3 expression in response to lipopolysaccharide, demonstrating that induction of SAA by recombinant hS100A4 protein was not due to endotoxin contamination. To examine the specificity of the induction of SAA expression by S100A4, we first tested the ability of different S100 family proteins to stimulate the accumulation of SAA in CM from VMR cells. In addition to S100A4, the proinflammatory S100A8 and S100A9 proteins also stimulated SAA expression, whereas S100A1, S100A2, S100A6, S100B, S100P and S100A12 had no effect (Figure 1d). The specificity was further confirmed by using two independent inhibitory anti-human S100A4 monoclonal antibodies that have no crossreactivity with mouse S100A4. They markedly inhibited hS100A4-mediated SAA expression, but not the induction of SAA Oncogene (2014) 1 – 12

expression by mouse recS100A4 (Figure 1e and Supplementary Figure S1D). In addition, mutant forms of hS100A4 (moA4mt1 and moA4mt2) that cannot form oligomers had an attenuated ability to induce SAA activation (Figure 1e and Supplementary Figure S1D). These data demonstrate that S100A4 specifically stimulates the expression of SAA in VMR cells. SAA augments the metastasis-associated properties of tumor cells in vitro We next examined whether SAA proteins modify tumor cell properties associated with metastasis. VMR cells were transduced with SAA1 and SAA3 retroviral expression vectors to obtain the stable SAA-expressing cell lines VMR/SAA1 and VMR/SAA3. Cells transduced with the empty vector (VMR/CTL) served as controls. Expression and secretion of SAA proteins was verified by western blotting (Supplementary Figure S2A). In subsequent experiments, the use of retrovirally transduced cells compared with exogenously added SAA protein (either as CM or as recombinant protein) allowed paracrine and autocrine effects to be compared. Both VMR/SAA1 and VMR/SAA3 cells adhered much more strongly to fibronectin than the VMR/CTL cells (Figure 2a), but not to laminin or collagen (Supplementary Figure S2B). Effects of SAA on cell motility were assessed by incubating wounded monolayers of mouse CSML100 and human MDA-MB-231 and SW480 tumor cells with CM from VMR/SAA1, VMR/SAA3 and VMR/CTL cells, or with medium supplemented with recSAAs. Effects on the motility of mouse embryonic fibroblasts were also examined, as S100A4 stimulates the motility of activated fibroblasts and recruits them to tumors, promoting metastasis.29 In all cases, SAA-containing media induced a significant increase in motility compared with control media (Figures 2b and c and Supplementary Figures S2C– E). Both recSAA1 and recSAA3 also exerted chemotactic effects in a dose-dependent manner on CSML100 tumor cells after 6 h in Transwell migration/invasion assays (Figure 2d). Significantly enhanced invasiveness of human MDA-MB-231 tumor cells was also observed in three-dimensional Matrigel assays that examined the effect of CM from VMR/SAA1 and VMR/SAA3 cells, as well as recSAAs compared with controls (Figure 2e and Supplementary Figure S2F). SAA proteins induce MMP expression in monocytic and endothelial cells.11,12 MMPs are implicated in metastatic progression. We therefore examined whether SAA proteins also upregulate MMP expression in VMR cells. Transcription of MMP2, MMP3, MMP9 and MMP13 was significantly increased in VMR cells treated with recSAAs (Figure 3a). Consistently, zymography demonstrated that CM from VMR/SAA1 and VMR/SAA3 cells contained increased proteolytic activity when compared with controls. The size of the cleared bands corresponded to MMP2 and MMP9 (Supplementary Figure S3A). Increased levels of MMP2, MMP3, MMP9 and MMP13 proteins were also detected in CM from VMR cells treated with SAA3 (Supplementary Figure S3B). Furthermore, CM taken from VMR cells treated with recS100A4 and recSAA3 proteins exhibited higher proteolytic activity in zymography assays (Supplementary Figure S3C), demonstrating direct effects of SAA proteins on protease production. Consistently, non-metastatic CSML0 cells that do not respond to recS100A4 by upregulating SAA transcription (Supplementary Figure S4A) showed no increase in proteolytic activity in response to S100A4 compared with VMR cells, but responded to recSAA (Supplementary Figure S4B). SAA proteins regulate the expression of a variety of cytokines.7,9,10 Furthermore, S100A4 induces the expression of RANTES (regulated upon activation normal T-cell expressed and presumably secreted) and G-CSF.28,30 We therefore examined whether SAAs induce the expression of cytokines and other proinflammatory proteins in VMR tumor cells. RecSAA1 or recSAA3 strongly stimulated transcription and accumulation of SAA & 2014 Macmillan Publishers Limited

S100A4-induced SAA expression promotes metastasis MT Hansen et al

3 Table 1.

Genes upregulated in VMR tumor cells in response to S100A4 (24 h) identified by RNA microarray analysis

Gene CXCL1 SAA3 EXPI ALDOA-PS1 PDZRN3 CEBPD LRIG1 SAA1 D19WSU12E PPAP2B CHI3L1 PDE4B MT1 D9UCLA1 HP SLPI SOD2 NFKB1 SCMH1 TRIM47 IER3 CYP7B1 BCL3 PIM1 RIN2 ADAMTS1 NFKB2 BID RELB PIK3AP1 ANXA1 IFNAR2 FTH1 ALDH3A2 CP GDNF MMP2 ATP2B4 FLNB AXL SMOX SOD2 FTH1 NADK S100A8 THRAP4 FTH1 COX7B TNIP1 ISP2 FTH1 LEPREL1 SHB RETNLG CTSL MT-ATP6 DHRS8 MT-ATP6 FAAH HIVEP1 OGFRL1 GLUL NEFH CSF1 GLIPR1 PSD PCOLN3 CCL4 MRPL17 DHX40

Fold induction 9.61836 3.09445 3.04594 2.88877 2.76743 2.14473 2.08822 1.91439 2.08696 1.93407 1.86881 1.76396 1.75631 1.75718 1.64029 1.62496 1.70735 1.6833 1.6032 1.57104 1.59972 1.52857 1.46493 1.57511 1.50839 1.54172 1.49329 1.47709 1.40966 1.43798 1.52158 1.48214 1.45364 1.44178 1.44929 1.39213 1.39512 1.46876 1.46582 1.42897 1.3777 1.40841 1.38935 1.5183 1.43334 1.43386 1.37284 1.43321 1.39607 1.33707 1.33253 1.44379 1.36273 1.36848 1.32786 1.32828 1.37775 1.32444 1.36722 1.36318 1.36524 1.31464 1.35809 1.30967 1.30676 1.34813 1.30149 1.28116 1.33649 1.33219

& 2014 Macmillan Publishers Limited

Description Cytokine CXCL1 Serum amyloid a-3 Extracellular proteinase inhibitor Aldolase A, fructose-bisphosphate-pseudogene PDZ domain containing RING-finger 3 Gene CCAAT/enhancer-binding protein (C/EBP), delta Leucine-rich repeats and Ig-like domains 1 Serum amyloid a-1 Endoplasmic reticulum metallopeptidase 1/ERMP1 Phosphatidic acid phosphatase type 2B Chitinase 3-like 1 gene Phosphodiesterase 4B, camp specific Metallothionein 1 HCV NS5A-transactivated protein 13 target protein 2 Haptoglobin Secretory leukocyte peptidase inhibitor Superoxide dismutase 2, mitochondrial Nuclear factor of kappa light polypeptide gene enhancer in B cells Sex comb on midleg homolog 1 Tripartite motif-containing 47 Immediate-early response 3 gene Cytochrome P450, family 7, subfamily b B-cell leukemia/lymphoma 3 Proviral integration site 1 Ras and Rab interactor 2 A disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 Nuclear factor of kappa light polypeptide gene enhancer in B cells 2, BH3-interacting domain death agonist Transcription factor (v-rel reticuloendotheliosis viral oncogene homolog B) Phosphoinositide-3 kinase adaptor protein 1 Annexin A1 Interferon (alpha and beta) receptor 2 Ferritin heavy chain 1 Aldehyde dehydrogenase family 3, subfamily A2 Ceruloplasmin Glial cell line-derived neurotrophic factor Matrix metallopeptidase 2 ATPase, Ca2 þ transporting, plasma membrane 4 Filamin, beta AXL receptor tyrosine kinase Spermine oxidase Superoxide dismutase 2, mitochondrial Ferritin heavy chain 1 NAD kinase S100 calcium-binding protein A8 (calgranulin A) Thyroid hormone receptor-associated protein 4 Ferritin heavy chain 1 Cytochrome c oxidase subunit viib TNFAIP3-interacting protein 1 Inplantation serine proteinase Ferritin heavy chain 1 Leprecan-like 1 Src homology 2 domain-containing transforming protein B Resistin-like gamma Cathepsin L Mitochondrially encoded ATP synthase 6 Estradiol 17-beta-dehydrogenase 11 Mitochondrially encoded ATP synthase 6 Fatty acid amide hydrolase Human immunodeficiency virus type I enhancer-binding protein 1 Opioid growth factor receptor-like 1 Glutamate-ammonia ligase (glutamine synthetase Neurofilament, heavy polypeptide Colony-stimulating factor 1 (macrophage) GLI pathogenesis-related 1 (glioma Pleckstrin and Sec7 domain containing Procollagen (type III) N-endopeptidase Chemokine (C–C motif) ligand 4 Mitochondrial ribosomal protein L17 DEAH (Asp-Glu-Ala-His) box polypeptide 40

Oncogene (2014) 1 – 12

S100A4-induced SAA expression promotes metastasis MT Hansen et al

4 Table 1.

(Continued )

Gene

Fold induction

Description

1.2775 1.32848 1.31799 1.27215 1.27223 1.25865 1.29413 1.26924

ETS2 PNRC1 NFE2L1 CYB5 PHC2 2810439K08RIK TAGLN2 FCNB

E26 avian leukemia oncogene 2, 3 domain Proline-rich nuclear receptor coactivator 1 Nuclear factor, erythroid-derived 2,-like 1 Cytochrome b-5 Polyhomeotic-like 2 (Drosophila) Hypothetical protein Transgelin 2 Ficolin B

The arrays were scanned by using an Axon GenePix 4000B microarray scanner (Axon Instruments Inc., Union City, CA, USA) and the GenePix Pro program (Biberach, Germany). Genes were considered as differentially expressed if they showed at least a 1.3-fold difference in signal intensity.

+S100A4 + -

+S100A4 + -

* **

5

+

** **

**

* 400

** * **

0

-

*

200

+

1.5

-

+

3 6 hours -

+

0

24

12

SW480

1.5

3

6

9

12

24

hours -

+

-

+

-

+hS100A4

+ SAA1

VMR

ns

0 0

-

Transactivation of SAA3 (x folds)

Transactivation of SAA1 (x folds)

15

10

SAA3

600

SAA1

SW620 MB-231 MB-435 MCF7

CTL

+Ab#1 +Ab#2 +IgG

SAA1 SAA3

MCF7s1

Tubulin Actin

recS100 +PMB

CTL

hA4

CTL

moA4

+Ab#1

+Ab#2

SAA1 CTL

A4

A8

A12

A6

S100B

A4

A8

S100P

A2

A1

A9

+Ab#1

+Ab#2

SAA3 CTL

A9

SAA1 CTL

SAA1

moA4 moA4mt1 moA4mt2

SAA1

Figure 1. S100A4 specifically upregulates SAA in VMR cells. SAA1 (A) and SAA3 (B) expression in VMR cells incubated with recS100A4 ( þ S100A4) for the indicated times was analyzed by qPCR, and normalized relative to non-treated controls (fold induction). Insets: Western blots of SAA1 and SAA3 in CM from VMR cells treated with ( þ ) and without (  ) recS100A4 for 24 h. The experiments were performed independently three times, each with three replicates for each time point. Representative western blot analyses are presented in the insets. (C) Western blot of SAA1 in CM from the indicated human cell lines treated with ( þ ) and without (  ) recS100A4 for 24 h. Loading control: actin. (D) SAA1 and SAA3 protein accumulation in CM from VMR cells incubated with the indicated recS100 proteins in the presence of PMB. (E) (a–c) Western blot of SAA in CM from VMR cells after stimulation with human S100A4 (hA4) in the presence of two different anti-human S100A4 monoclonal antibodies (Ab no. 1 and Ab no. 2) that inhibit SAA1 and SAA3 upregulation in response to hA4 but not mouse S100A4 (moA4). Non-treated cells (CTL) and isotype control antibodies (IgG) served as controls. Tubulin served as a loading control. (D) Mutated forms of moA4 (moA4mt1 and moA4mt2) unable to form oligomers and have an attenuated ability to induce SAA3. Representative western blot analysis is presented.

proteins in CM from VMR cells (Figures 3b and c). Moreover, RANTES, G-CSF, S100A8 and S100A9 were also transcriptionally upregulated in response to recSAA1 and recSAA3 (Figures 3d and e). Taken together, these data are consistent with the notion that SAA proteins produced by tumor cells in response to S100A4 are able to modulate cell adhesion, migration and invasion, to transcriptionally upregulate MMPs, cytokines and other proinflammatory factors, and to regulate positively their own expression (Figure 3f). Oncogene (2014) 1 – 12

Molecular regulation of SAA expression by S100A4 How does S100A4 induce SAA expression? TLR-4 can mediate S100A8 and S100A9 signaling31 via NF-kB and IRAK (interleukin-1 receptor-associated kinase).32 Specific inhibitors for TLR-4 (CLI-095), NF-kB (CAPE (caffeic acid phenethyl ester)) and IRAK (IRAK1/4 (interleukin-1 receptor-associated kinases 1/4 inhibitor)) robustly suppressed SAA accumulation in the CM from S100A4treated VMR cells (Figure 4a, Supplementary Figure S5A and Supplementary Figure S6), implicating these factors in the & 2014 Macmillan Publishers Limited

S100A4-induced SAA expression promotes metastasis MT Hansen et al

5 ***

6 4 2

cmVMR cmVMR/SAA1 cmVMR/SAA3

40

***

20

3

0

5

10

15 Hours

20

*** 80 ***

***

60 ***

40 cmVMR cmVMR/SAA1 cmVMR/SAA3

20 0

25

0

5

10 15 Hours

20

25

VM

R

/S

A

A A

CSML100

100 80

1 SA +r

/S

R VM

cm

A

3 A

A A /S VM

R

cm

A SA

A

1

R

60

VM

m

g/

5m 1

2m

m A

1

10

SA

g/

m

m g/

m A

3

3 A

SA

SA

g/

5m

C

2m 3 A SA

g/

m

l

TL

0

**

cm

100

**

*

120

R

200

140

VM

Relative area of invasion in matrigel (%)

Number of migrated cells

MDA-MB-231

***

300

cm

/C

/S

VM

R

R VM

60

A

1

0

*

80

100

Relative area of wound (%)

n.s.

MDA-MB-231

100

Relative area of wound (%)

8

TL

Adherent cells (fold induction)

CSML100

3

**

Transactivation (fold induction)

40

* ***

2

**

*

**

*

1 0 MMP2

50

CTL SAA1 SAA3

***

Transactivation of SAA (x folds)

4

CTL SAA1 SAA3

MMP3

MMP9

*** **

30 20 10 ** ** 0 RANTES

G-CSF

MMP13

Transactivation (fold induction)

Transactivation of MMPs (fold induction)

Figure 2. SAA proteins enhance tumor cell adhesion to fibronectin, stimulate migration and invasion, and upregulate MMPs. (a) Adhesion of VMR cells transduced with either empty vector (VMR/CTL), SAA1 (VMR/SAA1) or SAA3 (VMR/SAA3) retroviruses to fibronectin was quantified using crystal violet staining and absorbance (OD550). Values for mean absorbance minus uncoated wells±s.e.m. relative to VMR/CTL cells are plotted. One of two independent experiments is shown. (b and c) CM from VMR/SAA cell lines increases the motility of mouse CSML100 cells (b) and human MDA-MB-231 cells (c) in monolayer wound-healing assays. Wounded monolayers were incubated with CM from VMR cells transduced with either empty vector (VMR/CTL), SAA1 (VMR/SAA1) or SAA3 (VMR/SAA3) retroviruses. Wound closure was monitored for 22 h. (d) Chemotactic migration of CSML100 cells after 6 h incubation with the indicated recSAAs in Transwell migration assays. (e) SAA stimulates invasion of MDA-MB-231 cells in three-dimensional Matrigel assays. Assays were performed with either control CM (cmVMR), CM from VMR/ SAA1 cells (cmVMR/SAA1), CM from VMR/SAA3 cells (cmVMR/SAA3) or CM from VMR cells supplemented with recSAA1 (cmVMR þ SAA1). Each data point represents the mean of triplicate samples (b–e).

150

10 8

CTL SAA1 SAA3

**

4

*

SAA1

***

6

rS100A4 rSAA3 rSAA1 PMB -

***

2

+ -

+ +

+ -

+ +

+ -

0 SAA1

SAA3

S100A4

CTL SAA1 SAA3 ***

100

***

G-CSF RANTES

SAA

S100A8 S100A9

50 *** ** 0 S100A8

S100A9

MMPs

Figure 3. RecSAA proteins upregulate the expression of inflammation-associated genes. VMR cells were treated with recSAA1 or recSAA3. The experiments were performed independently three times, each with three or four replicate samples. (a) Expression of MMPs, (b) SAA1 and SAA3, (d) RANTES and G-CSF and (e) S100A8 and S100A9 in response was monitored by qPCR, and normalized (fold induction) relative to mock-treated cells (CTL). (c) Western blot of SAA1 in CM from VMR cells treated with recSAA1 and recSAA3. Representative western blot analysis is presented. Owing to its size, the tagged recSAA1 added where indicated to the CM lies outside the area of the gel presented in the figure. RecS100A4 served as a positive control. PMB treatment controlled for endotoxin contamination. (f ) Schematic diagram showing the S100A4-regulated expression of SAA, proinflammatory cytokines and MPPs, and the positively acting feedforward loops described in this paper. & 2014 Macmillan Publishers Limited

Oncogene (2014) 1 – 12

S100A4-induced SAA expression promotes metastasis MT Hansen et al

6 n.s.

Transactivation of SAA3 (fold induction)

50 SAA1 A4

-

+

-

LPS

-

-

+

Inhibitor

-

-

-

+

+

+

-

-

-

-

-

-

+

+

+

TLR4 IRAK NF-kB TLR4 IRAK NF-kB

**

40 30 20 10 0

SAA3

5 CTL TNF Luciferase transactivation (fold induction)

4

S100A4

S100A4

-

+

+

+

EGFR inh MEK inh

-

-

+ -

+

3 recSAA1 2

SAA1

1

0 VMR-CTL

VMR-I B dh-CI5

S100A4

-

+

-

-

-

-

-

-

SAA1

-

-

+

+

-

-

-

-

SAA3

-

-

-

-

+

+

+

+

PMB

-

-

-

+

-

+

-

-

Inhibitor

-

-

-

-

-

-

TLR4 NFkB

Figure 4. S100A4 and SAA signal via TLR4 and NF-kB. (a) VMR cells were treated with S100A4 (A4) or lipopolysaccharide in the presence and absence of TLR4, IRAK and NF-kB inhibitors. Expression of SAA1 in CM from the cells was assessed by western blotting. Representative western blot analysis from two independent experiments is presented. (b) VMR-I-kB and control cells (VMR-CTL) were engineered to express a NF-kBdriven luciferase reporter, and then were treated with S100A4 and tumor necrosis factor-a. Luciferase activity relative to mock-treated cells (CTL) was assessed. The experiments were performed independently three times, each with three replicate samples. (c) VMR cells were treated with S100A4 in the presence or absence of EGFR or MEK inhibitors. Expression of SAA3 relative to mock-treated cells was assessed by qPCR, and by western blot (insert). The experiments were performed independently four times, each with three replicate samples. A representative western blot is presented. (d) Western blots of SAA1 in CM from VMR cells treated with recS100A4, recSAA1 or recSAA3 in the presence and absence of PMB or inhibitors of TLR4 and NF-kB. Both the added recSAA1 and the endogenous cell-derived SAA1 are visible. A representative western blot analysis from two independent experiments is presented.

upregulation of SAA in response to S100A4. Epidermal growth factor (EGF)-induced extracellular signal-regulated kinase (ERK) phosphorylation was not affected by these inhibitors, verifying the specificity of the inhibition (Supplementary Figure S5B). Moreover, in VMR cells expressing a dominant-negative form of IkBa (VMRIkBa-cI5 cells), the activity of an NF-kB-responsive luciferase reporter in response to either S100A4 or tumor necrosis factor-a was significantly impaired compared with that in wild-type VMR cells (Figure 4b). As S100A4 modulates EGF receptor (EGFR) signaling,33 we analyzed whether this pathway is also involved in S100A4-driven activation of SAA3. VMR cells were incubated with the tyrosine kinase inhibitors AG1478 (EGFR-specific) and AG879 (ErbB2specific) before being treated with recS100A4. Inhibition of EGFR strongly and significantly suppressed S100A4-dependent SAA3 transcription and protein expression, whereas inhibition of the downstream kinase MEK did not influence S100A4-mediated upregulation of SAA3 mRNA (Figure 4c). Collectively, these data indicate a decisive role for NF-kB in S100A4-driven transcriptional activation of SAA genes, and suggest an involvement of TLR-4 and EGFR(s) in S100A4-mediated signal transduction. Although positive self-regulation by SAA also involves NF-kB, TLR-4 is not involved in this signaling pathway in VMR cells (Figure 4d). Ectopic SAA expression in VMR cells promotes tumor metastasis VMR-CTL and VMR-SAA cells were injected into experimental animals to determine whether SAA expression is sufficient to stimulate metastasis in vivo. Histological evaluation of tissue sections revealed that SAA3 expression resulted in significantly Oncogene (2014) 1 – 12

increased spontaneous and experimental metastases in both the lung and the liver (Figures 5a–d). Expression of SAA1 induced metastasis even more potently in the experimental metastasis assay (Figures 5e and f). In addition, SAA-overexpressing VMR cells instigated metastasis in other organs such as the spleen, lymph nodes, ovary, kidney and bones in 50% of the mice injected with VMR-SAA1 and in 80% of the mice injected with VMR-SAA3. A marked infiltration of CD45-positive leukocytes into the lungs and livers of animals bearing VMR-SAA tumors was observed, but not in animals bearing control VMR tumors (Figure 5g). Systemic S100A4 induces SAA expression and other transcriptional targets in an organ-specific manner Intravenous injection of S100A4 increases metastasis formation in the liver and lung by VMR cells.26 Furthermore, S100A8 induces SAA3 expression in the lung.14 We therefore examined whether increased systemic levels of proinflammatory S100A4, S100A8 or S100A9 proteins might facilitate metastasis by inducing the expression of SAA in these organs. Significantly upregulated expression of SAA1 in both the liver and the lung was observed after intravenous injection of S100A4 into mice for 2 weeks (Figures 6a and e). SAA3 was also significantly upregulated in the liver, but only tendentially in the lung (Figures 6b and f). S100A8 significantly induced SAA1 expression in the lung but only tendentially in the liver (Figures 6a and e), while SAA3 was upregulated in both the liver and the lung (Figures 6b and f). S100A9 on the other hand had no significant effect on SAA expression in any organ. Of the three proteins, S100A4 and S100A8 most potently induced SAA expression. In addition to & 2014 Macmillan Publishers Limited

S100A4-induced SAA expression promotes metastasis MT Hansen et al

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Figure 5. Ectopic SAA expression stimulates the metastatic potential of VMR tumor cells. VMR-SAA3 or VMR-CTL were used in spontaneous (a and b) and experimental (c and d) metastasis assays (nine mice per group). (e and f ) VMR-SAA1 was used in experimental metastasis assays (eight mice per group). Numbers of metastases in liver and lungs were evaluated. (g) Immunohistochemical staining with CD45 antibodies of sections of lung (upper panel) and liver (lower panel) from VMR-CTL and VMR-SAA3 tumor-bearing mice. Scale bars, 50 mm. Immunohistochemical stainings were repeated two times and representative data are presented.

inducing expression of SAA in these organs, S100A4 and S100A8 both induced an increase in the level of SAA3 protein in the blood of the treated animals (Figure 6i). Differences in the specific activity of the recS100A proteins can be ruled out, as all three preparations induced SAA3 expression in VMR cells (Figure 1d). In similar experiments, we also observed that S100A4 significantly increased the expression of RANTES, G-CSF, S100A8 and S100A9 in the liver (Figure 6c and d). In the lung, it also increased the expression of S100A9, downregulated G-CSF and had a negligible effect on RANTES and S100A8 (Figure 6g and h). These data demonstrate that S100A4 can regulate the expression of RANTES, G-CSF, S100A8 and S100A9 in vivo, but does so in an organ-specific manner. & 2014 Macmillan Publishers Limited

Coincident expression of SAA and S100A4 in human colon carcinomas is indicative of poor prognosis To determine the relevance of our findings to human cancer, we examined whether expression of SAA alone and in combination with S100A4 correlates with patient survival. Primary tumor specimens were analyzed from 60 colon adenocarcinoma patients whose tumors had not metastasized at the time of surgery. Of these patients, 23 subsequently developed distant metastases metachronously. SAA and S100A4 expression as determined by qPCR was independent of the UICC tumor stage, and the age and sex of the patients. There was no significant correlation between SAA expression and the metachronous development of metastases, although a trend of low SAA expression together with better overall survival was observed (Figure 7a). However, S100A4 Oncogene (2014) 1 – 12

S100A4-induced SAA expression promotes metastasis MT Hansen et al

S100A8

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8

SAA3

Figure 6. S100 proteins induce SAA and cytokine expression in an organ-specific manner. RecS100A4, recS100A8 and recS100A9 was injected intravenously into mice for 2 weeks. Expression of SAA1 (a and e), SAA3 (b and f ), RANTES and G-CSF (c and g), and S100A8 and S100A9 (d and h) in lungs, liver and spleen was then assessed using qPCR, relative to the mean of the PBS control (fold induction). (i) SAA3 protein was detected in western blots of serum prepared from animals treated with recS100A4, recS100A8 and recS100A9 proteins. Seven or eight mice were used in each group.

expression was higher in primary tumors that later developed distant metastases, and was significantly associated with reduced overall survival (Figure 7b). Importantly, high expression of both SAA and S100A4 was the best predictor of poor overall survival (Figure 7c). These findings are underscored by the 5-year survival rates, which showed substantially lower survival for patients with both high SAA and high S100A4 expression in their tumors compared with patients with elevated levels of only one of these factors, or with low expression of both (Table 2). Thus, S100A4 transcript levels in primary colon tumors predict overall survival, and this is further enhanced when combined with SAA expression. DISCUSSION Correlative studies with human tumors and functional experiments in animals link S100A4 expression with metastasis.34 The mechanism remains unresolved. Here we show that S100A4 stimulates expression of inflammation-associated genes in tumor cells in vitro and in non-transformed cells in vivo. Two of these genes, namely SAA1 and SAA3, profoundly influenced the metastatic properties of tumor cells in vitro and in vivo. Consistently, coordinate expression of S100A4 and SAA in human colorectal patient samples was indicative of poor prognosis. The data suggest that S100A4 stimulates metastasis through triggering an inflammatory response, mediated at least in part by SAA proteins. T-cell infiltration into both primary tumors and metastases is a feature of S100A4-mediated metastasis.28 The upregulation of inflammatory response proteins by S100A4, and the stimulation of metastasis by SAA we report here provide further evidence that S100A4 promotes metastasis at least in part by acting on the immune system. These inflammatory response proteins could promote metastasis in several ways. SAA induces neutrophilia,9 and tumor-associated neutrophils produce cytokines and Oncogene (2014) 1 – 12

chemokines that recruit and activate inflammatory cells, and promote tumor cell proliferation, angiogenesis and metastasis.35 Several cytokines produced in response to S100A4/SAA are involved in the recruitment of myeloid cells and regulatory T cells. Myeloid cells recruited to the tumor microenvironment suppress tumor immunity, and promote angiogenesis and metastasis.36 Regulatory T cells that infiltrate tumors can promote metastasis by secreting RANKL.37 Furthermore, the recruitment of myeloid cells is also a key event in the establishment of premetastatic niches,15 and S100A8 can induce SAA3 during premetastatic niche formation in the lungs.14 SAA proteins promoted MMP expression in tumor cells, and increased their motility and adhesion to fibronectin. Fibronectin accumulates in premetastatic lungs, and promotes metastasis formation.15 SAA-induced binding to fibronectin may therefore support recruitment of tumor cells to sites of metastasis formation. Binding of tumor cells to fibronectin also releases them from dormancy,38 suggesting that enhanced binding to fibronectin might promote outgrowth of metastases by suppressing quiescence. For the first time, we show that SAA proteins positively regulate their own expression, but in a TLR-4-independent manner in VMR cells. SAAs signal via several receptors, including TLR-2, TLR-4, RAGE, FPRL-1/ALX, SR-B1/ABCA1, CD55 and TANIS.4 Thus, a variety of alternative receptors other than TLR-4 could, in principle, be involved in self-regulated SAA expression. Expression of S100A8 and S100A9 was also stimulated by SAA, identifying a positive feedforward mechanism in which S100A4-mediated activation of SAA leads to self-amplifying accumulation of SAA and S100 proteins, and enhanced production of downstream targets such as metastasis-promoting cytokines and MMPs (Figure 3e). Thus, S100A4 produced by activated fibroblasts, macrophages and lymphocytes39,40 could trigger self-amplifying expression of SAA and S100 proteins in both primary tumors and metastatic sites, in turn promoting metastasis formation. This is a further example of & 2014 Macmillan Publishers Limited

S100A4-induced SAA expression promotes metastasis MT Hansen et al

9 SAA

Table 2. Five-year survival (5YS) rates of patients with primary colon adenocarcinomas correlated with the expression of SAA and S100A4

1.0

Overall survival based on SAA

0.8 low

5-Year survival (%)

±s.d.

Low High

89.3 71.9

0.058 0.079

S100A4

Low High

86.2 44.4

0.048 0.166

SAA þ S100A4

Both low SAA low S100A4 high SAA high S100A4 low Both high

92.0 66.7 80.8 33.3

0.054 0.272 0.077 0.192

Marker

Expression

SAA

0.6 high

0.4 0.2 0 0

50

100 Months

150

200

S100A4

The combination of both, SAA and S100A4, add benefit for prognostication. If both markers are low, the 5YS is 92% compared with 89% and 86% for SAA and S100A4, respectively. If both markers are high, the 5YS is 33% compared with 72% and 44% for SAA and S100A4, respectively. The most significant values are indicated in boldface.

1.0 P=0.001

Overall survival based on S100A4

0.8 low

0.6 0.4 high

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50

100 Months

150

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SAA and S100A4

Overall survival based on SAA and S100A4

1.0

P=0.007

0.8

both low

0.6

low S100A4, high SAA low SAA, high S100A4

0.4 both high

0.2 0 0

50

100 Months

150

200

Figure 7. Coincident expression of SAA and S100A4 expression in the primary tumors of patients with colon adenocarcinomas predict poor overall survival. SAA and S100A4 expression in the primary tumors of 60 patients with primary colon adenocarcinomas that had not metastasized at the time of surgery was determined by qPCR. Kaplan–Meier analyses for overall survival were performed for SAA expression (a), S100A4 expression (b) and coincident SAA and S100A4 expression (c). The survival time was set as the time difference between resection of the primary tumor and the last contact (censored data) or death.

positively acting feedforward loops that establish an inflammatory milieu and provide a potent stimulus to tumor growth at primary and secondary sites.34 & 2014 Macmillan Publishers Limited

Of the nine S100 family members tested, only the proinflammatory proteins S100A4, S100A8 and S100A9 were able to activate SAA expression in vitro in VMR tumor cells. In vivo, only S100A4 and S100A8 had any significant effect on SAA expression. Surprisingly, S100A12 is also a proinflammatory member of the S100 family, yet had no effect on SAA induction. These data suggest that the ability to promote metastasis formation through inducing expression of SAA proteins is restricted to only a few members of the S100 family. Ectopic SAA expression in VMR tumor cells markedly extended the range of organs in which VMR metastases were found. Consistently, mice bearing overt subcutaneous Lewis lung carcinoma tumors have been reported to exhibit enhanced metastatic colonization of the lung when SAA1-expressing Lewis lung carcinoma cells were additionally injected intravenously.41 Neither the effect of SAA1 on metastasis in non-tumor-primed mice, metastasis formation in organs other than the lung nor immune cell infiltration into the metastatic lesions was examined in this study. In contrast to the broad range of organs in which SAAexpressing tumor cells form metastases, the S100 proteins that induce SAA expression have a more organ-restricted effect on metastasis formation. In studies on Lewis lung carcinoma, transactivation of SAA3 by S100A8 but not S100A9 has been reported during premetastatic niche formation.14 Furthermore, metastasis of VMR cells to the liver and lung is increased when S100A4 is injected intravenously.26 As S100 family members interact with various receptors such as RAGE and TLR-4 to exert their effects,20 the ability of an individual S100 protein to regulate SAA expression is likely to be organ and cell-type dependent, and determined by the specific S100 receptor(s) expressed. We therefore suggest that the exact cells and organs in which SAA expression is induced by particular S100 proteins determines the organ specificity of metastasis formation by preconditioning these organs for metastatic growth. Moreover, we suggest that S100A4 expression either by tumor cells or stromal cells may be involved in premetastatic niche formation in the liver and in the lung.28 S100A8 has been reported to induce SAA3 expression in lungs but not liver.14 Here we observed that S100A8 significantly upregulated SAA3 in the liver, and only tendentially in the lung (Figure 6). There are several conceivable reasons for this discrepancy. The previously published study used ex vivo incubation of the organs with recS100A8,14 whereas our experiments were performed in vivo. Furthermore, we analyzed SAA3 expression using qPCR, whereas the other study used reverse transcription–polymerase chain reaction (RT–PCR). The in vivo experiments in the previous study used heparin-conjugated Oncogene (2014) 1 – 12

S100A4-induced SAA expression promotes metastasis MT Hansen et al

10 S100A8,14 whereas we used native protein. Mouse strain-specific differences may also be operative. The upregulation of genes involved in inflammation in response to S100A4 is consistent with our findings that S100A4 is involved in rheumatoid arthritis, psoriasis and idiopathic inflammatory myopathies.17,18 Interestingly, SAA expression is also strongly associated with rheumatoid arthritis,42 and induces angiogenesis, cytokine expression, leukocyte recruitment and MMP-mediated matrix degradation in this context by NF-kB signaling.43 In further work, we will determine whether S100A4 has a functional role in rheumatoid arthritis by inducing the expression of SAA proteins. In conclusion, SAA proteins produced in response to S100A4 have the potential to stimulate metastasis formation in several ways. As S100A4 and SAA exhibit several overlapping effects including activation of MMPs and cytokines, we suggest that the metastasis-promoting effects of S100A4 are mediated at least in part by the SAA proteins they induce. Our data are consistent with the notion that systemic effects of S100A4 transcriptional targets, most notably SAAs, can act to induce an inflammatory reaction in specific organs that prepares the organ microenvironment for metastasis formation. Future studies will focus on understanding how SAA acts on the immune system to promote metastasis formation, and whether SAA could serve as a therapeutic target for combating metastasis. MATERIALS AND METHODS Cell lines and reagents VMR, CSML0 and CSML-100 cells were cultured as described.21 Human breast (MB-MDA-231, MB-MDA-435, MCF-7 and MCF-s1) and colon cancer cell lines (SW480 and SW620) were obtained from the Danish Cancer Society’s biobank. Mouse embryonic fibroblasts, bone marrow macrophages and splenic T cells were isolated as described.2,9,44 Murine SAA1 and SAA3 cDNAs (ImaGenes, Berlin, Germany) were inserted into the pBABEpuro retroviral vector (Cell Biolabs Inc., San Diego, CA, USA). Retroviral transduction and selection of infected cells was performed as described.45 VMR-IkB clones were generated by transfection of VMR cells with a dominant-negative IkBa expression construct (kindly provided by Dr M Glukhova, Institute Curie, Paris, France). PMB was from (Gibco, Naerum, Denmark). AG1478, AG879, CAPE and the IRAK 1/4 were from (Sigma-Aldrich, Broendby, Denmark). The TLR-4 inhibitor CLI-095 was from Invitrogen (Taastrup, Denmark) and the MEK 1/2 inhibitor U0126 was from Promega (Stockholm, Sweden).

Microarray analysis RNA was prepared from snap-frozen cells using PeqGold according to the manufacturer’s instructions (PeqLab, Erlangen, Germany). The probe labeling, microarray chips, hybridization and scanning has been described previously.46

RNA purification and quantitative real-time PCR First-strand cDNA synthesis with RNA extracted using the Nucleospin Triprep kit (Macherey-Nagel, Du¨ren, Germany) was performed using SuperScript II RT (Invitrogen) with random primers, in duplicate or triplicate. Each qPCR (Fast SYBR Green Master Mix kit (Roche Applied Science, Indianapolis, IN, USA) in a LightCycler 2.0 instrument (Roche DiagnosticsA/S, Hvidovre, Denmark)) was run at least in duplicate.

Sequences of primer pairs used for mouse qPCR qPCR was carried out using the following primers: GAPDH (forward, 50 -CCA GCAAGGACACTGAGCAA-30 ; reverse, 50 -GGGATGGAAATTGTGAGGGA-30 ); mouse SAA1 (forward, 50 -AGTCTGGGCTGCTGAGAAAA-30 ; reverse, 50 -GGCA GTCCAGGAGGTCTGTA-30 ); mouse SAA3 (forward, 50 -ACATGTGGCGAGCCTA CTCT-30 ; reverse, 50 -GAGTCCTCTGCTCCATGTCC-30 ); MMP2 (forward, 50 -CAC ACCAGGTGAAGGATGTG-30 ; reverse, 50 -AGGGCTGCATTGCAAATATC-30 ); MMP9 (forward, 50 -TGAATCAGCTGGCTTTTGTG-30 ; reverse, 50 -ACCTTCCAGT AGGGGCAACT-30 ); MMP13 (forward, 50 -ACCCAGCCCTATCCCTTGAT-30 ; reverse, 50 -TTTGGGATGCTTAGGGTTGG-30 ); G-CSF (forward, 50 -CTCAACTTT CTGCCCAGAGG-30 ; reverse, 50 -TCCAGGGACTTAAGCAGGAA-30 ); RANTES (forward, 50 -CATATGGCTCGGACACCACT-30 ; reverse, 50 -ACACACTTGGCGGT Oncogene (2014) 1 – 12

TCCTTC-30 ); S100A4 (forward, 50 -TTGTGTCCACCTTCCACAAA-30 ; reverse, 50 GCTGTCCAAGTTGCTCATCA-30 ); S100A8 (forward, 50 -CCTTGCGATGGTGATA AAAGTG-30 ; reverse, 50 -CCCAGCCCTAGGCCAGAA-30 ); S100A9 (forward, 50 CAAAGGCTGTGGGAAGTAATTAAGA-30 ; reverse, 50 -AGCCATTCCCTTTAGACT TGGT-30 ).

Sequence of primers used for human qPCR For SAA and S100A4, amplicons of 153 and 124 bp were produced, respectively. The primers used were as follows: SAA (forward, 50 TGGTTTTCTGCTCCTTGGTC-30 ; reverse, 50 -CCCGAGCATGGAAGTATTTG-30 ) (SYBR green format); S100A4 (forward, 50 -CTCAGCGCTTCTTCTTTC-30 , primer, 50 -GGGTCAGCAGCTCCTTTA-30 ); FITC-labeled probe (50 -TGTGATGGT GTCCACCTTCCACAAGT-30 ); LCRed640-labeled probe (50 -TCGGGCAAAGAG GGTGACAAGT-30 ) (hybridization probes format).

Recombinant proteins and in vivo application Active oligomeric S100A4 protein was obtained as described.47 Murine cDNAs for SAA1 and SAA3 were inserted with in-frame V5-His tags into the bacterial expression vector pQE30 (Qiagen, Copenhagen, Denmark) and used to produce recombinant (recSAA) protein in Escherichia coli according to the manufacturer’s protocol. Human recSAA1 was purchased from Prospec (East Brunswick, NJ, USA). Tail vein injections and intradermal injections using rodent intradermal delivery devices (Becton Dickinson, Albertslund, Denmark) were performed on C57Bl/6 mice aged 8–12 weeks five times per week for 2 weeks (25 mg protein per injection). Phosphatebuffered saline-injected animals served as controls. Excised organs and serum were snap frozen.

Antibodies and western blotting Monoclonal anti-human S100A4 antibodies39 were used in 10-fold excess (10 mg/ml) over recS100A4 (incubation time 20 h) in S100A4 function inhibition experiments. Other antibodies used were rabbit anti-mouse SAA3 serum,30 rabbit anti-mouse SAA1 antibodies (a kind gift from Lars Bo Nielsen, Copenhagen University, København K, Denmark),48 mouse monoclonal anti-actin antibody AC-40 (Sigma-Aldrich), rabbit anti-MMP-3 antibodies (Epitomics, Burlingame, CA, USA) and rabbit anti-NF-kB p65 antibodies (Santa Cruz, Heidelberg, Germany). The high homology between SAA1 and SAA2 means that the SAA1 antibody also likely detects SAA2; therefore, we refer to the protein detected by this antibody as SAA1/2. Western blotting was performed using standard methods. Adhesion and motility assays. Adhesion assays and monolayer wound healing motility assays were performed as described.49,50 Relative migration was calculated as the mean area of wound remaining at a given time point relative to the original wound area. The threedimensional Matrigel invasion assay has been described.51 Chemotactic cell migration. Transwell plates with 12 inserts (pore size 8 mm) were coated with fibronectin. CSML100 cells (5  105 per well) in serum-free Dulbecco’s modified Eagle’s medium were added. Lower chambers contained serum-free media without or with recombinant proteins. After incubation at 37 1C, cells penetrating the insert membrane were stained with Diff-Quick (Dade Behring, Marburg, Germany). For each insert, migrated cells were counted in 20 independent microscopic fields. Two independent experiments were performed, each with triplicate samples.

Zymography Cells were grown to 90% confluence. The medium was exchanged with serum-free medium containing recombinant proteins. CM was harvested 6 or 24 h later, filtered through 0.45 mm membrane filters and concentrated using Vivapore or Vivaspin concentrators (Vivascience Ltd, Herlev, Denmark). The cells were subsequently trypsinized and counted to normalize the quantity of proteins in the CM used. The concentrated CM was assayed for protease activity using gelatin and casein zymography.52

Animal tumor experiments A/Sn mice aged 8–11 weeks were maintained according to European Laboratory Animal Science Association guidelines. Tumor cells (0.5  106 cells per mouse) were injected subcutaneously or intravenously. Experiments were terminated when the first animal became moribund. Organs & 2014 Macmillan Publishers Limited

S100A4-induced SAA expression promotes metastasis MT Hansen et al

11 were macroscopically assessed for the presence of metastases. Lungs and livers were analyzed using standard histology. Metastases per unit area were counted in tissue sections. Immunohistochemistry was performed using anti-CD45 antibodies (BD Pharmingen, San Diego, CA, USA).

Human tumor analysis Primary adenocarcinomas from 60 colon cancer patients were obtained after written consent and ethical approval, and snap frozen. All patients were untreated previously, did not have a history of familial colon cancer and underwent surgical resection at the Robert-Ro¨ssle Cancer Hospital, Berlin, Germany. None had metastases at the time of surgery, but 23 developed distant metastases metachronously. Tumor cells were microdissected from serial cryosections, and then total RNA was isolated. Duplicate qRT–PCR was performed as described previously.53 Calibrator cDNAs for the SAA-RT–PCR were derived from HepG2 cells, and for the S100A4-RT–PCR from HCT116 cells. These were used in serial dilutions simultaneously in each run.

Statistical analysis Metastatic burden comparisons used the Mann–Whitney test. P-values for the other analyses used the two-tailed unpaired Student’s t-test. P-values o0.05 were regarded as significant. *Po0.05, **Po0.01 and ***Po0.001. Statistical evaluation of human colorectal samples used the nonparametric two-sided Mann–Whitney rank-sum test. Kaplan–Meier curves were evaluated with the log-rank test. Cutoffs were calculated by receive operating characteristic analyses.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Ekaterina Dulina, Inge Skibshøj and Lene Bregnholt Larsen for careful technical assistance. We gratefully acknowledge funding by the European Union (TuMIC, Health-F2-2008-201662) and INARMERA (FP7-INCO-2010-6), the Danish Cancer Society and the Dansk Kræftforsknings Fond.

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