1 Tropomodulin 1 Expression Driven by NF-Κb Enhances Breast Cancer

Author Manuscript Published OnlineFirst on November 14, 2014; DOI: 10.1158/0008-5472.CAN-13-3455 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tropomodulin 1 expression driven by NF-κB enhances breast cancer growth

Taku Ito-Kureha1, 2, Naohiko Koshikawa3, Mizuki Yamamoto4, Kentaro Semba4, Noritaka Yamaguchi5, Tadashi Yamamoto2, Motoharu Seiki6, and Jun-ichiro Inoue1.

1Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 2Cell Signal Unit, Okinawa Institute of Science and Technology, Okinawa, Japan; 3Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 4Department of Life Science and Medical Bio-science, Waseda University, Tokyo, Japan; 5Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan; and 6Graduate School of Medicine, Kochi University, Kochi, Japan.

Running title: NF-κB-induced tropomodulin enhances breast tumor growth Keywords: NF-κB, tropomodulin, triple-negative breast cancer, MMP13, β-catenin

Funding: This work was supported by a grant-in-aid for Scientific Research on Innovative Areas and Scientific Research (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (J-i.I.).

Corresponding Author: Jun-ichiro Inoue, Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 813-5449-5275; Fax: 813-5449-5421; E-mail: [email protected]

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest are disclosed.

Word count: 5363 words Total number of figures and tables: 7 figures and no tables.

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 14, 2014; DOI: 10.1158/0008-5472.CAN-13-3455 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Triple-negative breast cancers (TNBC), which include the basal-like and claudin-low disease

subtypes, are aggressive malignancies for which effective therapeutic targets are lacking.

NF-κB activation has an established role in breast malignancy and it is higher in TNBC than

other breast cancer subtypes. On this basis, we hypothesized that proteins derived from

NF-κB target genes might be molecular targets for TNBC therapy. In this study, we

conducted a microarray-based screen for novel NF-κB inducible proteins as candidate

therapeutic targets, identifying tropomodulin 1 (TMOD1) as a lead candidate. TMOD1

expression was regulated directly by NF-κB and was significantly higher in TNBC than other

breast cancer subtypes. TMOD1 elevation associated with enhanced tumor growth in a

mouse tumor xenograft model and in a 3D type I collagen culture. TMOD1-dependent tumor

growth was correlated to MMP13 induction, which was mediated by TMOD1-dependent

accumulation of β-catenin. Overall, our study highlighted a novel TMOD1-mediated link

between NF-κB activation and MMP13 induction, which accounts in part for the

NF-κB-dependent malignant phenotype of triple-negative breast cancer.

Introduction

Gene expression analyses have defined five breast cancer subtypes (luminal-like,

ERBB2-enriched, basal-like, claudin-low, and normal-breast-like), each of which is thought

to be derived from a distinct differentiation stage of mammary epithelial cells, thereby

displaying unique prognostic features (1-3). The luminal-like subtype is characterized as

either an estrogen receptor positive (ER+) or a progesterone receptor-positive (PR+)

phenotype, whereas the basal-like and claudin-low subtypes constitute the majority of

triple-negative cancers (TNBCs; ER-, PR-, and ERBB2-negative), show a higher malignancy

than other subtypes and exhibit a poor prognosis against various methods of therapy. TNBCs

were identified based on low/absent expression of luminal differentiation markers, and

enrichment of epithelial-to-mesenchymal transition (EMT) and stem cell markers. Therefore,

while luminal-like cells appear more differentiated and form tight cell-cell junctions, TNBCs

appear less differentiated and have a more mesenchymal-like appearance. TNBCs are much

more frequently highly invasive (4) and show higher proliferative activity as reflected in high

Ki-67 expression when compared with luminal-like subtypes (5). Therefore, it is necessary to

ﬁnd molecular signatures and signaling pathways that contribute to the malignancy of

TNBCs.

The NF-κB family of transcriptional factors plays a critical role in inﬂammation,

immunoregulation, and cell differentiation (6, 7). This family consists of ﬁve members,

including p50, p52, RELA (p65), RELB, and c-REL, which form homomeric or heteromeric

dimers to activate transcription of the target genes. NF-κB is made transcriptionally inactive

by being sequestered in the cytoplasm when it forms complexes with the IκB family,

including IκBα, IκBβ, IκBε, and the p105 and p100 precursors of p50 and p52, respectively.

Nuclear translocation of NF-κB can be driven by two distinct signaling pathways. In the

canonical pathway, a large number of stimuli, including various cytokines and bacterial and

viral products, induce IκB kinase (IKK) β-catalyzed phosphorylation and proteasomal

degradation of IκBα, followed by nuclear translocation of mainly p50-RELA heterodimers

(8). The noncanonical pathway is activated by receptors that are crucial in the formation of

lymphoid organs and lymphocyte development, such as the lymphotoxin (LT) β receptor, the

receptor activator of NF-κB (RANK), and CD40. This pathway induces the IKKα-catalyzed

phosphorylation of the C-terminal half of p100 that sequesters RelB in the cytoplasm, which

leads to polyubiquitination-dependent processing of p100 to p52 and the nuclear

translocation of the p52-RELB heterodimers (9). Accumulating evidence indicates that

aberrant NF-κB activation leads to tumorigenesis and cancer malignancy through the

expression of genes involved in survival, metastasis, and angiogenesis (6, 10). We have

previously reported that TNBCs undergo constitutive and strong activation of NF-κB, whose

activation is transient in normal cells upon various physiological stimuli (11, 12).

Furthermore, the adenovirus-mediated expression of a nondegradable IκBα super-repressor

(IκBαSR), in which Ser-32 and -36 (the residues phosphorylated by IKKβ) were substituted

with alanine, blocked NF-κB activation, and thereby inhibited the growth of several TNBC

cell lines. This result strongly suggests that constitutive NF-κB activation plays a critical role

in TNBC malignancy.

In this report, we demonstrate that tropomodulin 1 (TMOD1) is a novel NF-κB

target gene and that TMOD1 protein, which inhibits the elongation and depolymerization of

actin filaments by binding to the pointed end of the actin filament (13-15), could be involved

in the enhancement of the in vivo growth of TNBC.

Materials and Methods

Cell culture

MDA-MB-436, MDA-MB-231 and BT-549 were obtained from the American Type Culture

Collection (ATCC), and resuscitated from early passage liquid nitrogen stocks as needed.

Cells were cultured for less than 3 months before reinitiating cultures and were routinely

inspected microscopically for stable phenotype.

Subcellular fractionation

Cells are suspended in Buffer A (10 mM HEPES-KOH pH 7.9 at 4°C, 1.5 mM MgCl2, 10

mM KC1, 0.5 mM dithiothreitol, 0.2 mM PMSF) and incubated for 15 minutes on ice

followed by centrifugation. The supernatant was stored as a cytoplasmic extract. The pellet

was resuspended in Buffer C (20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl,

1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF) and incubated on ice

for 20 min. Cellular debris was removed by centrifugation, and the supernatant was used as a

nuclear extract.

Tumor formation assay

MDA-MB-231 cells (106) in 100 μL of RPMI1640 (Wako) containing 50% Matrigel (BD

Biosciences) were subcutaneously injected into BALB/c Slc-nu/nu female mice (5-week-old,

Japan SLC, Inc.). Tumor growth was monitored twice a week. Animal research complied

with protocols approved by the Committee for Animal Experimentation of Waseda

University.

Chromatin immunoprecipitation (ChIP) assay

A ChIP assay was performed as described previously (12). Anti-RELA antibody (Santa Cruz)

was used for immunoprecipitation. The immunoprecipitation efficiency of the TMOD1

promoter region (-713 to -486) was analyzed by real-time PCR using FastStart Universal

SYBR Green Master (Roche). The primer set used was 5’-ATGTGGATCTGCTGCTTCCT-3’

and 5’- TTAAGCACCTACTGCATACA-3’.

Luciferase Assay

Cells were transfected with 1 μg of pTOPflash-Luc or pFOPflash-Luc and 0.001 μg of

pRL-Renilla Luciferase Reporter plasmid and harvested 48 h after transfection. Reporter

activities were measured with a luminometer (LB9507, Berthold) using a dual luciferase

reporter assay system (Promega). TCF/LEF activity was defined as the ratio of

TOPflash:FOPflash reporter activities.

Proliferation Assay

For 2D culture, cells (104/well) were cultured for 4 days, the viable-cell number was then

determined by trypan blue exclusion. For 3D culture, cells were suspended with chilled

collagen gel solution (Nitta Gelatin) (3 mg/mL) and seeded into 24-well plates (500 µl each,

5×103 cells/mL). After an 8-day culture, the cells were extracted from the collagen gels, and

the numbers of extracted cells were measured with a hemocytometer. In selected experiments,

cells were incubated with 5 μM MMI270 (kindly provided by Dr. M. Nakajima, Novartis

Pharma) or the recombinant MMP inhibitors, TIMP-1 (10 μg/mL) or TIMP-2 (5 μg/mL)

(Daiichi Fine Chemical Co., Ltd.).

Invasion assay

Transwell inserts (upper chamber) with 8 μm pore size were coated with collagen gel

solution and air-dried. Cells were added to each transwell. After 20 h, cells that had migrated

through the collagen gel and adhered to the other side of the insert were fixed and stained

with 0.5% crystal violet.

Microarray Analysis for identification of NF-κB target genes

The preparation of recombinant adenoviruses was performed as described previously (11).

RNA samples were prepared from MDA-MB-436 cells 24 hours after infections. Total RNA

labeled with Cy3- or Cy5- was hybridized to 3D-Gene Human Oligo chip 25k (25,370

distinct genes, Toray Industries Inc.). Genes with Cy3/Cy5 normalized ratios greater than 2.0

were defined. Microarray data have been deposited in the Gene Expression Omnibus (GEO)

under the accession number GSE56812.

Analysis of TMOD1 expression in breast cancers

DNA microarray analysis of 35 human breast cancer cell lines (CIBEX database accession no.

CBX20) (16) was used to analyze TMOD1 expression in human breast cancer cell lines. A

hierarchical clustering analysis of publicly available cDNA microarray data derived from

251 primary mammary tumor samples (GEO Series accession no. GSE3494) (17) was

performed using the GeneSpring software.

Statistical Analysis

Statistically significant differences between the mean values were determined using

Student’s t-test (***P < 0.001, **P < 0.01, and * P < 0.05). The values represent the means

of triplicate samples ± s.d.

Results

Screening of novel NF-κB target genes in TNBCs.

To search for novel NF-κB target genes involved in the malignancy of triple-negative breast

cancer, the claudin-low subtype cell line MDA-MB-436 was used because its NF-κB

activation level is significantly higher than those in other claudin-low cell lines (11). The

cells were infected with adenovirus expressing IκBαSR to block constitutive activation of

NF-κB (11, 18). To avoid the selection of genes whose expressions are nonspecifically

blocked by adenovirus infection, GFP-expressing adenovirus was employed (11). A

subsequent gene expression analysis using a DNA microarray led to the identification of 48

genes whose expressions were reduced more than 2-fold in IκBαSR-expressing cells when

compared with both mock- and GFP-infected cells (Fig. 1A and Supplementary Table S2).

Twenty-two known NF-κB target genes, including RELB, TNFAIP3 (also known as A20),

and BIRC3 (also known as cIAP2), were included in the 48 genes, indicating the validity of

our procedure. Among the remaining 26 genes, four genes, including tropomodulin 1

(TMOD1), chromosome 15 open reading frame 48 (C15orf48), cholesterol 25-hydroxylase

(CH25H), and vanin 2 (VNN2), were selected as candidates for novel NF-κB target genes

based on the semiquantitative RT-PCR analysis (Fig. 1B).

TMOD1 overexpression enhanced in vivo growth of MDA-MB-231 cells in the nude

mouse xenograft model.

To address the involvement of the four candidate genes in tumor progression in vivo, we used

the claudin-low subtype cell line MDA-MB-231 because this cell line can form tumors in the

xenograft model more efficiently than MDA-MB-436 and BT-549. Each gene was

introduced by gene transfer with the retrovirus vector, and then the population of cells that

stably expressed each gene was selected by puromycin treatment to generate 231-TMOD1,

231-C15orf48, 231-CH25H, and 231-VNN2. The control vector was used to generate

231-control (Fig. 2, A-D). Since infection efficiency was about 20% and no cell cloning, was

performed, the effects observed here are not limited to one specific cell clone, and it is also

unlikely that the selected population was biased compared to the parental cell population.

These cells were then injected bilaterally into the fat pads of BALB/c nu/nu mice, and tumor

growth was subsequently measured using calipers. Through this in vivo tumor growth

screening, we found that the overexpression of TMOD1 resulted in enhanced tumor growth

in nude mice, whereas the overexpression of the other three genes had no significant effect

(Figs. 2A-D). Tumors formed by injecting 231-TMOD1 appeared within a month, and the

TMOD1-overexpressing tumors were about two-fold larger compared with those of control

tumors 40 days after engraftment (Fig. 2A). In addition, tumor weights were also

significantly increased by TMOD1 overexpression when compared to control tumors (Fig.

2E). Histological analysis revealed that 231-TMOD1 tumors often had a necrotic area in their

central portion and a higher number of mitotic cells than 231-control tumors, but otherwise

showed no differences in morphology and angiogenesis (Fig. 2F). These data indicate that

TMOD1 is involved in promoting the in vivo growth of this claudin-low breast cancer cell

line.

TMOD1 gene expression was directly regulated by NF-κB, and TNBCs showed higher

TMOD1 expression than other breast cancers.

Whereas we found that TMOD1 expression was reduced by blocking NF-κB activation (Fig.

1B), it was unclear whether the transcription of TMOD1 was directly regulated by NF-κB.

We first investigated whether the TMOD1 expression was up-regulated by TNFα stimulation,

which induces NF-κB activation. Real-time RT-PCR analysis revealed that the expression of

TMOD1 mRNA was increased within the first 4 hours after TNFα stimulation in

MDA-MB-436 and MDA-MB-231 cells (Fig. 3A). Because the TMOD1 promoter has five

putative NF-κB-binding sites, we next analyzed the binding of RELA (also known as p65) to

the TMOD1 promoter region using a chromatin immunoprecipitation (ChIP) assay. RELA

was recruited to the TMOD1 promoter in both cell lines (Fig. 3B). Moreover, TMOD1

expression was also reduced by RELA down-regulation or treatment with Bay11-7082, an

inhibitor of IκBα phosphorylation, in various claudin-low cell lines (Fig. 3C and

Supplementary Fig. S1). These results indicated that the transcription of TMOD1 was

directly regulated by NF-κB.

Because NF-κB is highly activated in TNBCs and TMOD1 is an NF-κB target gene,

we next checked whether TMOD1 is preferentially expressed in TNBCs. Based on our DNA

microarray analysis of 35 human breast cancer cell lines (Supplementary Materials and

Methods) (16), TMOD1 expression is significantly higher in TNBCs (Fig. 3D). Nevertheless,

TMOD1 expression is varied among claudin-low cell lines used in this study (Supplementary

Fig. S1), which may account for their characteristic behavior. Furthermore, we used a

published dataset of human breast cancer specimens to investigate TMOD1 expression in

primary breast tumors. We first categorized 251 breast tumors (17) into basal-like,

claudin-low, ERBB2-enriched, and luminal-like subtypes based on a hierarchical clustering

analysis of their gene expression profiles (Fig. 3E, left). TMOD1 expression is significantly

higher in TNBCs than in ERBB2-enriched and luminal-like breast tumors (Fig. 3E, right). In

normal mouse mammary epithelial cells, Tmod1 expression is significantly higher in basal

and mature luminal cells than luminal progenitor cells (Fig. 3F). Because it has been reported

that luminal-like subtype tumors originate from mature luminal cells and basal-like subtype

tumors from luminal progenitor cells, high TMOD1 expression in TNBCs does not reflect

gene expression characteristics of normal epithelial cells. Together, these results point to

TMOD1 as a promising specific therapeutic target of TNBCs. TMOD1 was originally found

as an actin-capping protein that binds to the N-terminus of tropomyosin (19). Therefore,

pointed-end capping by TMOD1 helps to maintain the constant lengths of the actin filaments

in skeletal muscle and in the red cell membrane skeleton. However, the role of TMOD1 in

breast cancer development remains to be elucidated.

The TMOD1-induced enhancement of proliferation of MDA-MB-231 in the 3D culture

system is mediated by the MMP family

To elucidate the molecular mechanism of the TMOD1-dependent enhancement of in vivo

tumor growth (Fig. 2A), we first analyzed the effects of TMOD1 overexpression on cell

growth in a two-dimensional (2D) normal culture system. In 2D culture, 231-control and

231-TMOD1 showed similar rates of cell proliferation (Fig. 4A). In contrast, TMOD1

overexpression resulted in enhanced cell proliferation in the type I collagen 3D culture

system (Fig. 4B), a model culture system for in vivo tumor cell growth (20, 21). Because the

proportion of the sub-G1 population was not reduced by TMOD1 overexpression (Fig. 4C),

the enhancement of proliferation was due to enhanced cell division rather than reduced

apoptosis. Although less prominent than in the case of MDA-MB-231 cells, similar 3D

culture-specific and TMOD1 overexpression-dependent growth advantages were observed in

two other distinct claudin-low cell lines, MDA-MB-436 and BT-549 (Supplementary Fig.

S2A-C). These results suggest that TMOD1 overexpression could induce some protease

activities that degrade type I collagen. Consistent with this idea, 231-TMOD1 showed higher

invasive activity than 231-control (Fig. 4D). The invasive-growth capacity of malignant

cancer cells is linked to the abilities of cells to degrade extracellular matrix (ECM), and

members of the matrix metalloproteinase (MMP) family display specific proteolytic

activities against components of ECM (22, 23), which led us to hypothesize that some

members of the MMP family might be induced by the TMOD1 overexpression. To examine

involvement of the MMP family, we tested the effects of MMI270, an inhibitor for both

secreted and membrane-anchored MMPs (24), on the growth of 231-TMOD1 and

231-control in the 2D and 3D culture systems. In 3D culture, MMI270 treatment resulted in a

reduction in growth of both 231-control and 231-TMOD1 to similar levels (Fig. 4B), whereas

MMI270 had little effect on the growth of either in 2D culture (Fig. 4A). These results

strongly suggest that the TMOD1-induced enhancement of tumor cell proliferation in the 3D

culture system is mediated by MMP-dependent processes. To identify which members of the

MMP family were involved in the TMOD1-induced enhancement of 3D growth, we used

recombinant MMP inhibitors, TIMP1 and TIMP2: TIMP-1 is a potent inhibitor of secreted

MMPs but inefficiently inhibits membrane-anchored MMPs, whereas TIMP2 is an efficient

inhibitor of both membrane-anchored and secreted MMPs (25, 26). Both TIMP1 and TIMP2

significantly suppressed the growth rates of 231-TMOD1, bring it down to those of the

231-control in the 3D culture system (Fig. 4F), whereas both inhibitors had negligible effect

on the growth rates of MDA-MB-231 in the 2D culture system, irrespective of TMOD1

expression (Fig. 4E). These results indicate that both secreted and membrane-anchored types

of MMP are involved. Given that the ECM in the 3D culture is composed of type I collagen,

TMOD1 could induce expression of MMP members that can degrade collagen or gelatin, a

partial hydrolytic product of collagen.

MMP13 induction is likely involved in the TMOD1-mediated enhancement of 3D

growth.

To evaluate the function of endogenous TMOD1 in TNBCs, the effect of TMOD1

down-regulation on the 3D growth was tested. The effective knockdown of TMOD1 in

MDA-MB-231 cells was achieved using two distinct TMOD1 shRNAs (Fig. 5A). Although

TMOD1 down-regulation did not affect cell proliferation in 2D culture (Fig. 5B), the same

down-regulation resulted in reduced proliferation in the 3D culture (Fig. 5C). Since this

down-regulation did not induce apoptosis (Fig. 5D), the reduced rate of growth revealed that

TMOD1 is crucial for invasive growth within type I collagen gels.

Because the invasive growth of 231-TMOD1 was regulated in an MMP-dependent

manner (Figs. 4B and F), we investigated which MMPs caused the TMOD1-dependent

enhancement of the 3D cell growth. Semiquantitative RT-PCR experiments revealed that

TMOD1 down-regulation resulted in significant reduction of MMP13 mRNA expression,

whereas the expression of membrane-anchored type MMPs including MT1-MMP,

MT2-MMP and MT3-MMP and that of secreted type MMPs including MMP1, MMP2 and

MMP9 were all scarcely changed by TMOD1 down-regulation in MDA-MB-231 cell lines

(Fig. 5E). MMP13 mRNA was up-regulated by TMOD1 overexpression (Fig. 5F). MMP13

activity in the conditioned media was also regulated by TMOD1 expression (Fig. 5G and

Supplementary Figs. S3A). Similar TMOD1-dependent expression of MMP13 was also

observed in the claudin-low cell lines MDA-MB-439 and BT-549 (Supplementary Figs. S3B

and C). These results indicate that MMP13 is the downstream target of TMOD1.

Because we have not checked the effect of TMOD1 down-regulation or expression

on the MMP13 activity, we need to determine whether MMP13 contributes to

TMOD1-dependent 3D cell growth. Therefore, small interfering RNA (siRNA)-mediated

knockdown of MMP13 was conducted in 231-TMOD1 (Fig. 5H). In 3D culture, MMP13

down-regulation resulted in a reduction in the growth of both 231-TMOD1 and 231-control

to similar levels (Fig. 5J), whereas MMP13 down-regulation had negligible effect on the

growth of either cell line in 2D culture (Fig. 5I). Although relative levels of NF-κB activation

among the three claudin-low cell lines used in this paper (MDA-MB-231, 18.1;

MDA-MB-436, 48.8; and BT-549, 25.3, with NF-κB activity of TNFα-stimulated Jurkat

cells set to 100) (11) do not necessarily correlate with their relative expression levels of

TMOD1 and MMP13 (Supplementary Figs. S1 and S3D), NF-κB positively regulates

TMOD1 expression and TMOD1 positively regulates MMP13 expression in these cell lines

(Figs. 3A, 5E and F and Supplementary Figs. S3B and C). Thus, MMP13 could be a key

molecule in the NF-κB-dependent enhancement of 3D growth.

TMOD1-induced stabilization of β-catenin resulted in the upregulation of MMP13.

To understand the molecular mechanism by which TMOD1 induced expression of MMP13,

we checked the effects of TMOD1 down-regulation on the mitogen-activated protein kinase

(MAPK) and β-catenin pathways in MDA-MB-231 cells because these pathways are

involved in MMP13 expression (27-30). TMOD1 down-regulation had little effect on the

phosphorylation of ERK, JNK, and p38 (Fig. 6A). However, the amounts of β-catenin

protein, but not mRNA, in TMOD1 knockdown cells were significantly decreased (Fig. 6A

and B). Because β-catenin is normally found in the cytoplasm but is found in the nucleus

when activated (31), the subcellular localization of β-catenin was analyzed in the TMOD1

knockdown cells. The amounts of nuclear β-catenin were significantly reduced by TMOD1

knockdown using two distinct shRNAs, whereas those of cytoplasmic β-catenin were only

slightly decreased (Fig. 6C). In contrast, both nuclear and cytoplasmic β-catenin was

up-regulated in 231-TMOD1 (Fig. 6D). Furthermore, when TMOD1 down-regulation cells

were treated with MG132, the levels of β-catenin were restored to those of MG132-treated

control cells (Fig. 6E). These results strongly suggest that TMOD1 can protect β-catenin

from its proteasomal degradation, thereby stabilizing β-catenin protein. Consistent with this,

the β-catenin-driven transcriptional activity of the TOPFLASH-FOPFLASH reporter system

declined by 50% when TMOD1 was down-regulated (Fig. 6F). Similar TMOD1-dependent

stabilization of β-catenin was observed in other claudin-low cell lines (Supplementary Fig.

S4A and B). Together, these results show that TMOD1 was associated with the activation of

the β-catenin/TCF-Lef pathway, which led to the induction of MMP13.

Discussion

In this study, we identified a novel NF-κB target gene, TMOD1, which is highly expressed in

TNBCs, including basal-like and claudin-low breast cancers. The overexpression of TMOD1

in the claudin-low breast cancer cell line MDA-MB-231 resulted in enhanced tumor growth

in a mouse xenograft model. In addition, a series of in vitro experiments strongly suggested

that TMOD1 is likely to stabilize β-catenin, which then induces one of the β-catenin target

genes, MMP13, whose down-regulation significantly blocked the TMOD1-dependent

enhancement of proliferation in the 3D type I collagen culture. It has been reported that

membrane-anchored type MMPs are crucial for cancer cells to grow in ECM in vivo (32),

while secreted type MMPs that are first generated as an inactive proform and become

activated through processing by the membrane-anchored type, are also crucial for invasive

growth (33). Although expression of MMP1, MMP2 and MMP9 was little affected, that of

MMP13, which is activated by MT1-MMP (34), was significantly reduced by TMOD1

down-regulation. Because cross-linked collagens surround and confine the cells, proteolysis

of collagen into gelatin by membrane-anchored type MMPs generates certain interstices and

allows cells to grow. Given that MMP13 degrades both type I collagen and gelatin (35), the

enhanced MMP13 expression induced by the elevated expression of TMOD1 likely results in

the generation of more interstices than those generated by MT1-MMP alone. Therefore, the

enhanced expression of TMOD1 due to constitutive NF-κB activation could lead to the

enhanced proliferation of claudin-low breast tumor in vivo (Fig. 7). In this scenario,

MT1-MMP activity must be needed for MMP13 to enhance growth. In fact, TIMP2, which

inhibits both MT1-MMP and MMP13, suppressed the 3D growth of MDA-MB-231 more

efficiently than TIMP1, an inhibitor for MMP13. Consistent with this, MT1-MMP and

MMP13 are highly expressed in various breast cancers and regulate their bone metastasis (36,

37).

MMP13 is known to be induced by the β-catenin/LEF1 transcription complex

through the LEF-1-binding site in the MMP13 promoter (30). Our studies revealed that

TMOD1 down-regulation resulted in a reduction in β-catenin whereas TMOD1

overexpression augmented β-catenin. Therefore, TMOD1 overexpression leads to enhanced

MMP13 expression. It has been reported that Wnt-β-catenin signaling is involved in the

malignancy of the basal-like subtype of breast cancer (38, 39) and that its activation is

enriched in basal-like breast cancers and predicts a poor outcome (40). TMOD1 expression

could be involved in the activation of the Wnt-β-catenin pathway in vivo. While the

downstream targets of the β-catenin pathway, MYC and cyclin D1, are clearly relevant in

tumor formation because of their role in proliferation, apoptosis, and cell-cycle progression

(41, 42), VEGF-A and MMPs were also downstream targets (30, 43-45). Therefore, our data

suggest that TMOD1 is likely to be associated with the promotion of angiogenesis in addition

to the degradation of ECM, which together are involved in metastasis.

Although the mechanism underlying the TMOD1-mediated increase in β-catenin

remains to be elucidated, our results suggest that TMOD1 inhibits the constitutive

degradation of β-catenin by proteasomes. Adenomatous polyposis coli (APC) is a gene

responsible for the onset of familial adenomatous polyposis (FAP), an autosomal dominantly

inherited disease that predisposes patients to multiple colorectal polyps and cancers. Because

APC can induce the degradation of β-catenin, the inactivation of APC results in the

stabilization and accumulation of β-catenin, thereby leading to tumor formation. In contrast,

APC, together with Asef or IQGAP1, regulates formation of the actin meshwork. Given that

TMOD1 is an actin-capping protein, TMOD1 may indirectly inhibit APC, which leads to the

stabilization of β-catenin followed by induction of the Wnt target gene MMP13 (Fig. 7).

Further studies are required to elucidate the mechanism of TMOD1-induced β-catenin

accumulation. In addition to MMP13 induction, the stabilization of the actin filaments by the

TMOD1-mediated capping of the pointed end of actin may enhance the growth of breast

cancer cells in the 3D culture system.

In this paper, we not only identify TMOD1 as a novel NF-κB target gene in

claudin-low breast cancer cells but also find that up-regulation of TMOD1 protein induced

by constitutive NF-κB activation results in nuclear accumulation of β-catenin, which in turn

could induce in vivo tumor growth by up-regulation of the β-catenin target gene MMP13.

Although further investigations are required, this NF-κB-TMOD1-β-catenin-MMP13 axis

could be involved in malignant phenotypes of other tumors. Because NF-κB and β-catenin

are ubiquitously expressed and their activation is required for various important cellular

functions, their inhibition would likely exert profound side effects. TMOD1, a linker

between NF-κB and β-catenin, could be a new therapeutic target for curing malignant breast

cancers.

Acknowledgments

We thank K. Miyazaki and T. Seki for assistance.

Authors’ Contributions

Conception and design: T.I. Kureha, N. Koshikawa, N. Yamaguchi, J. Inoue

Development of methodology: T.I. Kureha, N. Koshikawa, M. Yamamoto

Acquisition of data (provided animals, acquired and managed patients, provided

facilities, etc.): T.I. Kureha, N. Koshikawa, T. Yamamoto

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,

computational analysis): T.I. Kureha, N. Koshikawa,

Writing, review, and/or revision of the manuscript: T.I. Kureha, N. Koshikawa, J. Inoue

Administrative, technical, or material support (i.e., reporting or organizing data,

constructing databases): N. Koshikawa, M. Yamamoto, M. Seiki

Study supervision: M. Seiki, J. Inoue

References

1. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, et al. Repeated observation

of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A.

2003;100:8418-23.

2. Vargo-Gogola T, Rosen JM. Modelling breast cancer: one size does not fit all. Nat Rev

Cancer. 2007;7:659-72.

3. Herschkowitz JI, Simin K, Weigman VJ, Mikaelian I, Usary J, Hu Z, et al. Identification of

conserved gene expression features between murine mammary carcinoma models and human

breast tumors. Genome Biol. 2007;8:R76.

4. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast

cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell.

2006;10:515-27.

5. Aleskandarany MA, Green AR, Benhasouna AA, Barros FF, Neal K, Reis-Filho JS, et al.

Prognostic value of proliferation assay in the luminal, HER2-positive, and triple-negative

biologic classes of breast cancer. Breast Cancer Res. 2012;14:R3.

6. Inoue J, Gohda J, Akiyama T, Semba K. NF-κB activation in development and progression

of cancer. Cancer Sci. 2007;98:268-74.

7. Hayden MS, Ghosh S. Shared principles in NF-κB signaling. Cell. 2008;132:344-62.

8. Hacker H, Karin M. Regulation and function of IKK and IKK-related kinases. Sci STKE.

2006;2006:re13.

9. Sun SC. Controlling the fate of NIK: a central stage in noncanonical NF-κB signaling. Sci

Signal. 2010;3:pe18.

10. Karin M. Nuclear factor-κB in cancer development and progression. Nature.

2006;441:431-6.

11. Yamaguchi N, Ito T, Azuma S, Ito E, Honma R, Yanagisawa Y, et al. Constitutive

activation of nuclear factor-κB is preferentially involved in the proliferation of basal-like

subtype breast cancer cell lines. Cancer Sci. 2009;100:1668-74.

12. Yamamoto M, Ito T, Shimizu T, Ishida T, Semba K, Watanabe S, et al. Epigenetic

alteration of the NF-κB-inducing kinase (NIK) gene is involved in enhanced NIK expression

in basal-like breast cancer. Cancer Sci. 2010;101:2391-7.

13. Kostyukova AS. Tropomodulins and tropomodulin/tropomyosin interactions. Cell Mol

Life Sci. 2008;65:563-9.

14. Gregorio CC, Weber A, Bondad M, Pennise CR, Fowler VM. Requirement of

pointed-end capping by tropomodulin to maintain actin filament length in embryonic chick

cardiac myocytes. Nature. 1995;377:83-6.

15. Tsukada T, Kotlyanskaya L, Huynh R, Desai B, Novak SM, Kajava AV, et al.

Identification of residues within tropomodulin-1 responsible for its localization at the pointed

ends of the actin filaments in cardiac myocytes. J Biol Chem. 2011;286:2194-204.

16. Ito E, Honma R, Yanagisawa Y, Imai J, Azuma S, Oyama T, et al. Novel clusters of highly

expressed genes accompany genomic amplification in breast cancers. FEBS Lett.

2007;581:3909-14.

17. Miller LD, Smeds J, George J, Vega VB, Vergara L, Ploner A, et al. An expression

signature for p53 status in human breast cancer predicts mutation status, transcriptional

effects, and patient survival. Proc Natl Acad Sci U S A. 2005;102:13550-5.

18. Traenckner EB, Pahl HL, Henkel T, Schmidt KN, Wilk S, Baeuerle PA. Phosphorylation

of human IκBα on serines 32 and 36 controls IκBα proteolysis and NF-κB activation in

response to diverse stimuli. EMBO J. 1995;14:2876-83.

19. Fowler VM. Identification and purification of a novel Mr 43,000 tropomyosin-binding

protein from human erythrocyte membranes. J Biol Chem. 1987;262:12792-800.

20. Yamada KM, Cukierman E. Modeling tissue morphogenesis and cancer in 3D. Cell.

2007;130:601-10.

21. Debnath J, Brugge JS. Modelling glandular epithelial cancers in three-dimensional

cultures. Nat Rev Cancer. 2005;5:675-88.

22. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor

microenvironment. Cell. 2010;141:52-67.

23. Lemaitre V, D'Armiento J. Matrix metalloproteinases in development and disease. Birth

Defects Res C Embryo Today. 2006;78:1-10.

24. Kasaoka T, Nishiyama H, Okada M, Nakajima M. Matrix metalloproteinase inhibitor,

MMI270 (CGS27023A) inhibited hematogenic metastasis of B16 melanoma cells in both

experimental and spontaneous metastasis models. Clin Exp Metastasis. 2008;25:827-34.

25. Hotary KB, Yana I, Sabeh F, Li XY, Holmbeck K, Birkedal-Hansen H, et al. Matrix

metalloproteinases (MMPs) regulate fibrin-invasive activity via MT1-MMP-dependent and

-independent processes. J Exp Med. 2002;195:295-308.

26. Nagase H, Woessner JF, Jr. Matrix metalloproteinases. J Biol Chem. 1999;274:21491-4.

27. Balbin M, Pendas AM, Uria JA, Jimenez MG, Freije JP, Lopez-Otin C. Expression and

regulation of collagenase-3 (MMP-13) in human malignant tumors. APMIS.

1999;107:45-53.

28. Borden P, Solymar D, Sucharczuk A, Lindman B, Cannon P, Heller RA. Cytokine control

of interstitial collagenase and collagenase-3 gene expression in human chondrocytes. J Biol

Chem. 1996;271:23577-81.

29. Uria JA, Jimenez MG, Balbin M, Freije JM, Lopez-Otin C. Differential effects of

transforming growth factor-β on the expression of collagenase-1 and collagenase-3 in human

fibroblasts. J Biol Chem. 1998;273:9769-77.

30. Yun K, Im SH. Transcriptional regulation of MMP13 by Lef1 in chondrocytes. Biochem

Biophys Res Commun. 2007;364:1009-14.

31. Nathke I. Cytoskeleton out of the cupboard: colon cancer and cytoskeletal changes

induced by loss of APC. Nat Rev Cancer. 2006;6:967-74.

32. Itoh Y, Seiki M. MT1-MMP: a potent modifier of pericellular microenvironment. J Cell

Physiol. 2006;206:1-8.

33. Taniwaki K, Fukamachi H, Komori K, Ohtake Y, Nonaka T, Sakamoto T, et al.

Stroma-derived matrix metalloproteinase (MMP)-2 promotes membrane type

1-MMP-dependent tumor growth in mice. Cancer Res. 2007;67:4311-9.

34. Yana I, Seiki M. MT-MMPs play pivotal roles in cancer dissemination. Clin Exp

Metastasis. 2002;19:209-15.

35. Knauper V, Will H, Lopez-Otin C, Smith B, Atkinson SJ, Stanton H, et al. Cellular

mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that MT1-MMP

(MMP-14) and gelatinase a (MMP-2) are able to generate active enzyme. J Biol Chem.

1996;271:17124-31.

36. Chang HJ, Yang MJ, Yang YH, Hou MF, Hsueh EJ, Lin SR. MMP13 is potentially a new

tumor marker for breast cancer diagnosis. Oncol Rep. 2009;22:1119-27.

37. Figueira RC, Gomes LR, Neto JS, Silva FC, Silva ID, Sogayar MC. Correlation between

MMPs and their inhibitors in breast cancer tumor tissue specimens and in cell lines with

different metastatic potential. BMC Cancer. 2009;9:20.

38. Hatsell S, Rowlands T, Hiremath M, Cowin P. β-catenin and Tcfs in mammary

development and cancer. J Mammary Gland Biol Neoplasia. 2003;8:145-58.

39. Brennan KR, Brown AM. Wnt proteins in mammary development and cancer. J

Mammary Gland Biol Neoplasia. 2004;9:119-31.

40. Khramtsov AI, Khramtsova GF, Tretiakova M, Huo D, Olopade OI, Goss KH.

Wnt/β-catenin pathway activation is enriched in basal-like breast cancers and predicts poor

outcome. Am J Pathol. 2010;176:2911-20.

41. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, et al. Identification of

c-MYC as a target of the APC pathway. Science. 1998;281:1509-12.

42. Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M, Pestell R, et al. The cyclin

D1 gene is a target of the β-catenin/LEF-1 pathway. Proc Natl Acad Sci U S A.

1999;96:5522-7.

43. Easwaran V, Lee SH, Inge L, Guo L, Goldbeck C, Garrett E, et al. β-Catenin regulates

vascular endothelial growth factor expression in colon cancer. Cancer Res. 2003;63:3145-53.

44. Crawford HC, Fingleton BM, Rudolph-Owen LA, Goss KJ, Rubinfeld B, Polakis P, et al.

The metalloproteinase matrilysin is a target of β-catenin transactivation in intestinal tumors.

Oncogene. 1999;18:2883-91.

45. Brabletz T, Jung A, Dag S, Hlubek F, Kirchner T. β-catenin regulates the expression of

the matrix metalloproteinase-7 in human colorectal cancer. Am J Pathol. 1999;155:1033-8.

Figure legends

Figure 1. Screening for a novel NF-κB target gene. A, The scheme for screening novel

NF-κB-target genes. B, Semiquantitative RT-PCR to analyze the effects of IκBαSR

expression on the expression of the candidate genes.

Figure 2. TMOD1 enhances tumor growth in a mouse xenograft model. A-D, Control

MDA-MB-231 cells (231-control) and the cells expressing one of the four candidate genes

(231-TMOD1, -C15orf48, -CH25H, or -VNN2) were subcutaneously implanted into the fat

pads of nude mice. n: Number of injections. Expression of each candidate protein was

determined by western blotting using the anti-FLAG antibody. E, Tumors formed in mice

(left). Scale bars, 10 mm. Weights of tumors 40 days after injection (right). F, Sections of

tumors from mice injected with 231-control or 231-TMOD1 were stained with haematoxylin

and eosin. N denotes a necrotic area. Arrowheads indicate mitotic cells. Scale bars, 100 μm

(upper) and 50 μm (lower).

Figure 3. TMOD1 expression is directly regulated by NF-κB. A, The amounts of TMOD1

mRNA were measured by RT-PCR (upper). The bands were quantified by densitometry

(lower). Expression levels of 18S rRNA were used for normalization. B, A schematic of the

TMOD1 promoter highlighting the region used in the ChIP assay (upper). The primers used

(arrows) and putative NF-κB binding sites (closed rectangles) are indicated. The amounts of

precipitated DNA relative to the input DNA were determined by real-time RT-PCR (lower).

C, After RELA siRNA transfection, TMOD1 and RELA expression was measured by

real-time RT-PCR and immunoblotting. D, The TMOD1 mRNA expression in each subtype

is shown using a box and whisker plot, which is depicted with boxes showing the median and

the 25th and 75th percentile, with whiskers showing the minimal and maximal values. E,

Hierarchical cluster analysis of the published gene expression profiles of 251 human primary

breast tumors performed with GeneSpring software (left). Each cluster was classified based

on the expression of subtype-specific genes. The bottom colored bar indicates the tumor

subtypes. The TMOD1 mRNA levels among primary breast cancers are shown (right) as in

(D). F, Expression levels of Keratin 14 (Krt14), Keratin 18 (Krt18), estrogen receptor 1

(Esr1) and TMOD1 mRNA were measured by real-time RT-PCR. Those of β-actin mRNA

were used for normalization. The Sorting procedure for various mouse mammary epithelial

cell populations (left) and confirmation of each population (upper right) were shown.

Figure 4. TMOD1 enhances tumor proliferation in the type I collagen-mediated 3D culture.

A and E, 231-control and 231-TMOD1 cells were seeded at an initial density of 104 cells/well

in the presence or absence of MMI270 (A), TIMP1 or TIMP2 (E). Viable cells were counted

4 days after initial seeding and then seeded again at the same density. After a 4-day culture

with or without inhibitors, viable cells were counted. The total numbers of viable cells

derived from the initial cultures were calculated. B and F, 231-control and 231-TMOD1 cells

were seeded at an initial density of 5 × 103 cells/well in type I collagen gels in the presence or

absence of MMI270 (B), TIMP1 or TIMP2 (E). After an 8-day culture, viable cells were

counted. C, Nuclei were stained with propidium iodide, and DNA contents were measured

with a flow cytometer. D, Invasiveness was determined with transwell chambers using 10%

FCS as a chemoattractant.

Figure 5. MMP13 is involved in the TMOD1-mediated enhancement of tumor proliferation.

A, Knockdown of TMOD1 protein in MDA-MB-231 cells. B and C, Parental MDA-MB-231

cells and those expressing various shRNAs were seeded at an initial density of 104 cells/well

in 2D culture (B) or 5 × 103 cells/well in type I collagen gels (C). Further experiments were

performed as described in Figure 4 without MMP inhibitors. D, Nuclei were stained with

propidium iodide, and DNA contents were measured with a flow cytometer. E, The

expression levels of various members of the MMP family were analyzed by semiquantitative

RT-PCR. F, MMP13 expression was measured by real-time RT-PCR. G, MMP13 activity in

the condition media was analyzed by casein zymography. H, Knockdown of MMP13 in

MDA-MB-231 cells after siRNA transfection. I and J, After MMP13 siRNA transfection, cell

growth assays in the 2D (I) and 3D (J) culture conditions were performed as described in (B)

and (C).

Figure 6. TMOD1 activates the β-catenin/TCF-Lef pathway. A, The expression levels of

β-catenin, pERK, pJNK, and p-p38 were analyzed by western blotting. B, Real-time RT-PCR

analysis of β-catenin mRNA. C and D, Subcellular localization of β-catenin. The effects of

TMOD1 down-regulation (C) and TMOD1 overexpression (D) on the subcellular

localization of β-catenin were analyzed by subcellular fractionation followed by western

blotting. E, Western blot analysis of β-catenin in the cells cultured in the presence or absence

of the proteasomal inhibitor MG-132 (10 μM) for 12 h (upper). The bands were quantified by

densitometry (lower). The expression levels of β-catenin were normalized to those of

α-tubulin. Fold changes were calculated relative to the amount of β-catenin in parental cells

in the absence of MG132. F, TOPFLASH or FOPFLASH reporter plasmids were transfected

into 231-shTm1-1 and 231-shLuc cells. TCF-Lef activities were defined as the ratio of

TOPFLASH/FOPFLASH reporter activities. Data are shown as the fold induction relative to

the values obtained in the 231-shLuc cells.

Figure 7. A model illustrating how the NF-κB-dependent induction of TMOD1 enhances the

invasive growth of malignant TNBCs.

A B

IκBα-SR vs Mock IκBα-SR vs GFP MDA-MB-436 Virus: Mock GFP IκBα-SR TMOD1

48385 432 C15orf48

CH25H

VNN2

RelB

18S RNA 22 genes : known NF-κB target genes 26 genes : semi-quantitative RT-PCR to check their NF-κB-dependent expression

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2014 American Association for Cancer Research. Figure 1 Author Manuscript Published OnlineFirst on November 14, 2014; DOI: 10.1158/0008-5472.CAN-13-3455 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A B 231-control (n=6) 600 231-TMOD1 (n=8) 600 231-control (n=4) ) )

** 3 3 ** 231-C15orf48 (n=4)

400 231-control 231-TMOD1 400 TMOD1 231-control 231-C15orf48 * C15orf48 Tubulin 200 ** 200 Tubulin * * * * Tumor volume (mm volume Tumor Tumor volume (mm volume Tumor 0 0 10 20 30 40 day 10 20 30 40 day E C 0.6 600 231-control 231-TMOD1 **

) 231-control (n=4) 0.5 3 231-CH25H (n=4) 0.4 400 0.3 231-control 231-CH25H 0.2 CH25H 0.1 200 Tubulin (g) weight Tumor 0 Tumor volume (mm volume Tumor 0 10 20 30 40 day 231-control 231-TMOD1

D 231-control (n=4) F 231-control 231-TMOD1 600 231-VNN2 (n=4) ) 3 NN 400 231-control 231-VNN2 VNN2 Tubulin 200 Tumor volume (mm volume Tumor 0 10 20 30 40 day

MDA-MB-231 MDA-MB-231 MDA-MB-436 -1000 -717-607-589 -394 -38 +1 0.4 *** TNFα: 04 12 240 4 12 24 h 0.15 0.3 ** *** TMOD1 ** 0.2 0.1 18S rRNA 0.1 6

TMOD1 / 18SrRNA / TMOD1 0 5 0.05

4 (%) / input ChIP 3 siRNA: 0 control RELA-1 RELA-2 2 ol 1 Antibody : r RELA Fold change Fold ont RELA RELA 0 c control Tubulin

DA-MB-436 D E M MDA-MB-231

** 8 * ** * 4 n.s * 6

expression ESR1 3 PGR 4 K8 expression K18 GATA3 2 ERBB2 2 FOXC1 TMOD1

K5 TMOD1 K14 1 EGFR 0 CDH1 Basal Luminal CLDN3 CLDN4 0

CLDN7 in

Relative -like -like OCLN Relative inal

n=14 n=21 like

Basal iched ike - Ratio (log2 scale) -low Claud Luminal-like ERBB2-enriched Lum

ERBB2 -l

-enr Basal-like Claudin-low -4.40 4.4 F 67.7% Krt14 Krt18 Esr1 Luminal progenitor 1.2 (LP) cells K14–K18+ER + 0.8

CD61 0.4 CD61 Mature luminal (ML) cells 0 CD49f 66.7% LP ML B LP ML B LP ML B K14–K18+ER + expression Relative CD49f 2 1.5 CD24

92.4% expression 1 Basal (B) cells

CD49f Relative CD24 K14+K18–ER – 0.5 TMOD1

0 CD49f LP ML B

Figure 3 A D 4 0

) 8 day d day 8 4 45 40 6 * 35 30 25 4 20 15 2

10 Relativeinvasion 5

Total cell number (×10 number cell Total 0 0 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 MMI270 : – +

4 8 B E 0

y y

) a a 4 18 ) *** day d d 4 60 16 *** *** 14 50 12 40 10 8 30 6 n.s 20 4 10 2 Total cell number (×10 number cell Total 0 (×10 number cell Total 0 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 MMI270 : – + – TIMP1 TIMP2

C F 16 ) *** 4 100 14 *** 80 12 10 60 8 n.s N.S 40 6 n.s Sub G1 (%) G1 Sub 20 4 0 2 Total cell number (×10 number cell Total 0 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 – TIMP1 TIMP2

Figure 4 A B C ) ) 4 4

4 y y 8 14 *** MDA-MB-231 ay 0 80 d da da 12 *** 60 10 8 Virus : 40 Mock shLuc shTm1-1 shTm1-2 6 4 TMOD1 20 2

Tubulin (×10 number cell Total Total cell number (×10 number cell Total 0 0 Virus : Mock shLuc shTm1-1 shTm1-2 Virus : Mock shLuc shTm1-1shTm1-2 MDA-MB-231 MDA-MB-231

l D 100 H 80 siRNA: -3 60 E contro 13-1 13-2 13 40 N.S MDA-MB-231 Sub G1 (%) G1 Sub 20 0 231-control 231-control 231-control 231-control Virus : 231-TMOD1 231-TMOD1 231-TMOD1 231-TMOD1 Mock shLuc shTm1-1 shTm1-2 MT1-MMP MMP13 MT2-MMP Tubulin

I 0 4 )

MT3-MMP 4

231-shLuc 231-shTm1-1 231-shTm1-2 60 y 8

a MMP1 50 day day d F MMP2 40 1.2 ** MMP9 1 30 0.8 MMP13 0.6 TMOD1 20 0.4 18S rRNA 10 0.2 0 (×10 number cell Total 0 MMP13 / 18SrRNA MMP13 / 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 siRNA : None Control 13-1 13-2 13-3 G Conditioned media ) J 4 16 *** 14 *** 12 10 n.s 8 n.s WB:MMP13 231-control 231-control 231-TMOD1 231-shTm1-1 231-shLuc n.s Zymo. (+Ca 2+ ) kDa 6 Pro-MMP13 4 Intermediate-MMP13 50 Active-MMP13 2

C-terminal inactive 37.5 (×10 number cell Total 0 fragment Zymo. (+EDTA) 50 37.5

SDS-PAGE 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 231-control 231-TMOD1 (CBB stain) siRNA : None Control 13-1 13-2 13-3

Figure 5 E C A β -catenin β Tubulin PARP1 Virus : -catenin Tubulin Virus : Fold change MG132: 0 1 2 3 β pERK1/2 Tubulin -catenin p-p38 pJNK Virus : Mock Cytoplasmic Mock extract shLuc shLuc –

Mock MDA-MB-231 MDA-MB-231 shTm1-1 MDA-MB-231 shTm1-1 shLuc shTm1-2 shTm1-2 s Mock hTm1-1 Mock shLu shTm1-2

c Nuclear extract + shLuc shTm1-1 shTm1-1 shTm1-2 shTm1-2 D B F Virus : β β -catenin /18SRNA -catenin Tubulin PARP1 0 0.2 0.4 0.6 0.8 1.0 1.2

Virus : TOPFLASH / FOPFLASH 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Cytoplasmic Mock extract MDA-MB-231 shLuc 231-co MDA-MB-231 ntrol shLuc

*** 231-TMOD shTm1-1 Figure 6 1 shT m1-1 Nuclear Nuclear extract 231-co ntrol shTm1 231-TMOD -2 1 inactivation APC TMOD1 degradation TMOD1 Asef TMOD1 Actin filaments β-catenin β-catenin MT1-MMP Collagen digestion TMOD1 NF-κB mRNA nuclear accumulation invasive activation growth MMP13 β-catenin mRNA secretion Gelatin digestion Pro-MMP13 MMP13

Triple Negative Breast Cancer Cell Figure 7 Author Manuscript Published OnlineFirst on November 14, 2014; DOI: 10.1158/0008-5472.CAN-13-3455 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tropomodulin 1 expression driven by NF-κB enhances breast cancer growth

Taku Ito-Kureha, Naohiko Koshikawa, Mizuki Yamamoto, et al.

Cancer Res Published OnlineFirst November 14, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-13-3455

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2014/11/15/0008-5472.CAN-13-3455.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2014/11/14/0008-5472.CAN-13-3455. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.