C-Myb Enhances Breast Cancer Invasion and Metastasis Through the Wnt/Β

Author Manuscript Published OnlineFirst on April 5, 2016; DOI: 10.1158/0008-5472.CAN-15-2302 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

c-Myb enhances breast cancer invasion and metastasis through the Wnt/β-

catenin/Axin2 pathway

Yihao Li2, Ke Jin1, Gabi W. van Pelt3, Hans van Dam2,6, Xiao Yu4, Wilma E. Mesker3, Peter

ten Dijke1,2,6, Fangfang Zhou2,5*, Long Zhang1,2*

1 Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang

University, Hangzhou 310058, China

2Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands,

3Department of Surgery, 4Department of Anatomy and Embryology, Leiden University

Medical Center, Leiden, The Netherlands;

5Institutes of Biology and Medical Science, Soochow University, Suzhou 215123, PR China;

6Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Box

595, 75124, Uppsala, Sweden

Running title: c-Myb potentiates Wnt signaling in breast cancer

*Address correspondence to:

Long Zhang, Life Sciences Institute and Innovation Center for Cell Signaling Network,

Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, China. Tel: +86-571-

88208375; email: [email protected];

Fangfang Zhou, Institutes of Biology and Medical Science, Soochow University, Suzhou

215123, PR China. Tel: +86-512-65882491; email: [email protected].

There no potential conflicts of interest to be disclosed

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 5, 2016; DOI: 10.1158/0008-5472.CAN-15-2302 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

The molecular underpinnings of aggressive breast cancers remain mainly obscure. Here we

demonstrate that activation of the transcription factor c-Myb is required for the pro-metastatic

character of basal breast cancers. An analysis of breast cancer patients led us to identify c-

Myb as an activator of Wnt/β-catenin signaling. c-Myb interacted with the intracellular Wnt

effector β-catenin and co-activated the Wnt/β-catenin target genes Cyclin D1 and Axin2.

Moreover, c-Myb controlled metastasis in an Axin2 dependent manner. Expression

microarray analyses revealed a positive association between Axin2 and c-Myb, a target of the

pro-inflammatory cytokine IL-1β that was found to be required for IL-1β-induced breast

cancer cell invasion. Overall, our results identified c-Myb as a promoter of breast cancer

invasion and metastasis through its ability to activate Wnt/β-catenin/Axin2 signaling.

Key words: c-Myb, Wnt/ β-catenin signaling, Axin2, breast cancer progression.

Introduction

Increased expression of transcription factor c-Myb is associated with malignant human

cancers(1). In colorectal cancer, c-Myb has been reported to cooperate with activated β-

catenin to activate the c-Myc promoter and enhance c-Myc expression (2). In addition, c-

Myb overexpression, amplification and dysregulation has been detected in some subtypes of

breast cancer (3). In ER+ cells, c-Myb is induced by estrogen/ER signaling, required for

tumorigenesis and inhibits apoptosis in primary tumor cells (4,5). c-Myb is also involved in

the transforming growth factor (TGF)-β-induced epithelial-to-mesenchymal transition (EMT)

process (6). In addition, increasing evidence indicates that c-Myb is found in invasive breast

cancer associated with poor prognosis (7). However, the regulation of c-Myb activity and its

downstream signaling networks in these processes remain largely unclear.

Wnt proteins are secreted cytokines that play pivotal roles in development, cancer and other

diseases. Canonical Wnt/β-catenin signaling is initiated when Wnt ligands interact with a

heterodimeric transmembrane receptor complex, which consists of Frizzled (Fz) plus a low

density lipoprotein receptor-related protein (LRP) (8). Upon Wnt binding the receptor

complex, LRP is phosphorylated, which triggers disruption of the β-catenin ‘destruction

complex’. This enables β-catenin to translocate to the nucleus and form a complex with

TCF/LEF (T-cell specific transcription factor/lymphoid enhancer-binding factor) that drives

Wnt-induced gene expression (9). In breast cancer, Wnt-induced signaling is a promoter in

EMT and invasion/metastasis (10), and components of the canonical Wnt signaling pathway

have been identified as important mediators of breast cancer metastasis and as markers of

poor prognosis (11). A number of Wnt target genes have been linked to oncogenesis,

including Cyclin D1 and Axin2. Activation of Cyclin D1 is associated with poor clinical

outcome (12). Axin2 is a Wnt suppressor and mediates negative feedback. However, Axin2

also has tumor-promoting functions; it can for example trigger an EMT-like process in breast

cancer cells via stabilization of Snail (13). In the current study, we demonstrate a mechanistic

link between c-Myb and Wnt/β-catenin signaling in basal breast cancer cells, which plays an

important role in invasion and metastasis.

Materials and Methods

Cell culture and reagents

Human MDA-MB-231-luc (BM) cells were obtained as previously mentioned (14).

HEK293T and MDA-MB-231 cells were provided by ATCC. 4T1 and MCF10A-M2 cells

were a kind gift from Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI,

USA). The 4T1 cells were used within 20 passages. The human cell lines have been

authenticated by short tandem repeat (STR) profiling within 6 months of the last experiment.

4T1, HEK293T and MDA-MB-231 cells are cultured in DMEM (high glucose with L-

glutamine) supplemented with 10% FBS and 100 U/ml Pen/Strep (Gibco). The MCF10A-M2

cells were maintained as previous described (15). All cell lines were maintained at 37°C with

5% CO2.

Immunoblotting and immunoprecipitation

Immunoprecipitationand western blotting was performed as previous described (16). Actin or

Tubulin protein level were used as loading control. We used the following primary antibodies:

anti-N-cadherin (610920, BD Transduction Laboratories), anti-Slug (9585, Cell Signaling),

anti-β-catenin (610154, BD Transduction Laboratories), anti-c-Myb (12319s, Cell Signaling),

anti-LEF-1(2230, Cell Signaling), anti-Axin2 (2151 Cell Signaling), anti-tubulin (2148, Cell

Signaling), anti-HA (1583816, Roche), anti-Flag (F3165, Sigma) and anti-β-actin (A5441,

Sigma). All secondary antibodies were from Sigma.

RNA isolation and real-time qPCR

RNA was isolated using the NucleoSpin RNA II kit (BIOKE) followed by reverse

transcription PCR (RevertAid First Strand cDNA Synthesis Kits, Fermentas). The cDNAs

were analysed by real-time quantitative PCR (qPCR), and calculations were performed using

the CFX Manager software version 2.0 (Bio-Rad). All mRNA expression levels were

analysed in triplicate and normalized to GAPDH expression. The primer sequences are listed

in supplemental material and methods.

Experimental animals

The zebrafish embryonic invasion and metastasis assay was performed as described

previously (17). All the zebrafish data are representative of at least two independent

experiments and performed according to the guidelines for use of laboratory animals in

Leiden University.

Mice experiments were approved by the Committee for Animal Welfare in Zhejiang

University. For the intracardial injection assay, five-weeks-old female BALB/c nude mice

were anesthetized with isofluorane and 1 × 105 freshly cells were injected into the left heart

ventricle. 5 weeks after injection, all mice were sacrificed and metastasis were analysed. The

mouse mammary fat pad implantation was based on previously published method (18).

Female BALB/c mice were anesthetized and used for this assay. 2×105 4T1 cells were

injected through the nipple area into the mammary fat pad. At 21 days after injection, primary

tumor were analysed. Then the mice were euthanized and analysed for secondary tumor. All

primary/metastatic tumors were detected by BLI with the IVIS 100 (Caliper Life Sciences,

Hopkinton, MA, USA). The BLI signal intensity was quantified as the sum of photons within

a region of interest given as the total flux (photons/second).

Statistical analysis

The results are expressed as the mean ± s.d. (standard deviation). For analysis, we used

Student's t-test and p<0.05 was considered to be statistically significant. ∗: 0.01

0.001

zebrafish embryos displaying tumor progression. The correlation between genes expression

were analysed by Pearson’s coefficient tests. For analysing TMA results. The χ² test was used

to calculate correlation between c-Myb and Axin2.

Results

c-Myb is critical for pro-metastatic traits in breast cancer cells

To investigate the function of c-Myb in breast cancer progression, we first analysed copy

number alterations of c-Myb in the TCGA Research Network (http://cancergenome.nih.gov)

by cBioportal (19,20). c-Myb is amplified in more than 2% of breast cancer patients, while 15%

show c-Myb gain (Supplementary Fig. S1A). Notably, c-Myb amplification and gain is

associated with a poor clinical outcome (Supplementary Fig. S1B). Oncomine plots show that

c-Myb is highly expressed in both invasive ductal and lobular breast cancer, suggesting that

c-Myb may play a role in tumor progression (Fig. 1A) (20-23). In addition, elevated c-Myb

expression has also been observed in ER- breast cancers, which are frequently invasive and

metastatic (24).

To study the function of c-Myb in breast cancer progression, which is associated with

enhanced breast cancer migration and invasion (25), we silenced endogenous c-Myb

expression in two invasive basal breast cancer cell lines, MDA-MB-231 and MCF10A-M2, in

which c-Myb protein was readily detected. Figure 1B showed the c-Myb knockdown

efficiency in these cell lines at both mRNA and protein levels. Depletion of c-Myb resulted in

a significant decrease in cell invasiveness in a transwell assay in both cell lines (Fig. 1C). In

line with the putative tumor-promoting effect of c-Myb, we found that the mRNA levels of

various mesenchymal markers, including Twist, Snail and Slug, were decreased in these cells

after c-Myb knockdown (Fig. 1D). Immunoblotting showed decreased protein expression of

N-cadherin, Slug and Snail in c-Myb-depleted cells (Fig. 1E). In addition, there was a

positive correlation between the mRNA expression of c-Myb and mesenchymal makers like

N-Cadherin, Snail and Slug in 189 breast cancer patients in a publicly available microarray

database (GSE2990) (26) (Fig. 1F). We also examined the prognosis of the patients from this

particular dataset and found that high expression of c-Myb is associated with a poorer clinical

outcome (Fig. 1G). These clinical results are consistent with the experimental data indicating

that c-Myb has a promoting role in breast cancer progression.

c-Myb regulates breast tumor progression in zebrafish xenografts

We next tested the role of c-Myb on malignant breast cancer in vivo by using a previously

published zebrafish embryo xenograft model (15), in which injected red-fluorescence

labelled tumor cells can circulate in the blood vessels, extravasate from circulation and

eventually metastasize into the tail fins (Fig. 2A). Embryos transplanted with c-Myb-depleted

MDA-MB-231 cells showed much less tumor progression than embryos injected with control

cells (Fig. 2B and C). At all time points of the experiment, both the average invasive area

and the percentages of embryos exhibiting metastasis were significantly lower in the two

groups injected with c-Myb-depleted cells (Fig. 2D and E). Compared to MDA-MB-231,

MCF10A-M2 cells show moderate metastatic potential in this zebrafish model and form a

cluster of cells between the dorsal aorta and the caudal vein (17). Ectopic expression of c-

Myb in MCF10A-M2 cells enhanced migration in vitro (Supplementary Fig. S2A and B), in

line with the pro-invasive function of c-Myb revealed in Figure 1C. In the zebrafish xenograft

model forced c-Myb expression in MCF10A-M2 cells resulted in increased metastasis and a

larger area of invasion (Fig. 2F-I). When the control MCF10A-M2 cells were implanted, 24%

of the embryos showed tumor mass formation at 6 dpi. Ectopic expression of c-Myb

increased this percentage nearly two-fold (45% of embryos), further confirming the positive

role of c-Myb in breast cancer progression (Fig. 2J).

c-Myb activates Wnt/β-catenin target genes and forms a complex with β-catenin/LEF-1

in breast cancer

We next investigated which signaling networks act at downstream of c-Myb to modulate

breast cancer progression. For this we analysed the association between c-Myb and candidate

signaling pathways, including the TGF-β, Wnt, NFκΒ and Notch pathways, using the TCGA

Research Network (data not shown) (20). We observed a significant positive correlation

between c-Myb and three metastasis-associated direct targets of Wnt, namely Axin2, LEF-1

and Cyclin D1 among 534 breast cancer patients (Fig. 3A) (27). Consistent with this notion,

ectopic c-Myb expression in HEK293T cells consistently and significantly increased the

transcriptional activity of the canonical Wnt reporter TOPFlash-Luc in a dose-dependent

manner (Fig. 3B). We subsequently investigated the possibility that c-Myb could function as

an activator of the Wnt/β-catenin pathway during breast cancer progression. In line with this

hypothesis the mRNA levels of the Wnt target genes Axin2 and Cyclin D1 were remarkably

decreased in c-Myb deficient MDA-MB-231 and MCF10A-M2 cells upon stimulation with

recombinant Wnt3a (100ng/ml) (Fig. 3C). These results suggested that c-Myb is required for

the expression of Wnt target genes in these basal breast cancer cells.

The activation of transcription by canonical Wnt signaling is triggered by the binding of

nuclear β-catenin to TCF/LEF transcription factors on target promoters. We previously

reported that c-Myb can interact with LEF-1 in leukemic cells (28). Interestingly, in HeLa

and Cos-7 cells ectopic c-Myb co-localized in the nucleus with a degradation-resistant β-

catenin mutant (SA) (Supplementary Fig. S3). Moreover, the ectopically expressed HA-c-

Myb was found to strongly interact with Flag-β-catenin in HEK293T cells (Fig. 3D). Co-

immunoprecipitation experiments in MDA-MB-231 and MCF10A-M2 cells also showed an

interaction between endogenous c-Myb and β-catenin (Fig. 3E). These findings suggested

that c-Myb can activate canonical Wnt target genes by binding to β-catenin and/or LEF-1.

Subsequent analysis of the binding of c-Myb to progressive deletion mutants of β-catenin

showed that the amino-terminal activating arm (NTAA) of the armadillo repeat (ARM)

domain of β-catenin (3-6 repeats) is essential for the interaction (Fig. 3F). This ARM motif is

important for the binding of β-catenin co-factors involved in transcriptional activation of Wnt

target genes, and in addition for TCF/LEF binding (4–10 repeats) (29). Importantly, we found

that the interaction between β-catenin and LEF-1 was reduced when c-Myb was knocked

down in MDA-MB-231 cells (Fig. 3G). Moreover, chromatin immunoprecipitations showed

that depletion of c-Myb inhibited the recruitment of β-catenin to the promoters of Axin2 and

Cyclin D1 (Fig. 3H). Taken together, these data suggest that c-Myb acts as a β-catenin co-

factor that stabilizes its interaction with TCF/LEF and thereby can enhance Wnt signaling-

induced transcription.

c-Myb promotes breast tumor progression through Axin2

As a direct target gene of Wnt/β-catenin, Axin2 not only mediates negative feedback , but

also can enhance EMT and cancer progression (13). In line with the RNA data in Figure 3,

the Axin2 protein level was strongly decreased in c-Myb-depleted MDA-MB-231 cells (Fig.

4A). We therefore examined the role of Axin2 in c-Myb-induced breast cancer

invasion/metastasis. For this, we depleted Axin2 in both normal MDA-MB-231 cells and

MDA-MB-231 cells ectopically overexpressing c-Myb, in which the endogenous Axin2

protein levels are increased by c-Myb (Fig. 4B). Figure 4B also shows that Axin2 expression

was efficiently blocked by Axin2-specific shRNA. These MDA-MB-231 cells were

subsequently injected into the duct of Cuvier of zebrafish to analyse their metastatic potential

(Fig. 4C and D). Compared to the embryos injected with the control cells, the embryos

implanted with c-Myb-overexpressing cells showed a higher tumor area and an increased

percentage of tumor progression to the tail fin (70% vs. 53% in controls) (Fig. 4E and F).

Moreover, the depletion of Axin2 significantly repressed invasion and metastasis in both the

control MDA cells and the c-Myb overexpressing cells (Fig. 4E and F). To confirm that

Axin2 is required for the c-Myb mediated breast cancer progression, we ectopically

expressed Axin2 in c-Myb depleted MDA-MB-231 cells (Supplementary Fig. S4A). The

ectopic expression could partly restore tumor progression in the zebrafish xenografts

(Supplementary Fig. S4B-D). Thus, the c-Myb-induced zebrafish metastasis of these basal

breast cancer cells is dependent on Axin2.

Because Axin2 turned out to be a critical target gene of c-Myb in breast cancer cell invasion,

we further analysed the mechanism by which c-Myb activates Axin2 gene expression. Since

c-Myb is a sequence specific DNA binding protein, we examined whether it can bind and

regulate the Axin2 promoter directly. Ectopic expression of c-Myb could significantly induce

Axin2 promoter-driven luciferase reporter in HEK293T cells (Fig. 4G) (30). We identified

three potential c-Myb binding motifs (TAACt/gG) in the Axin2 promoter at positions -923, -

728 and -614 (31). Next, we performed chromatin immunoprecipitations to specifically

analyse the binding of c-Myb to the region encompassing, the two proximal c-Myb binding

sites (c-Myb binding 1, -745 to -598), and the region encompassing the more distal site (c-

Myb binding 2 ,-959 to -858) (Fig. 4H). Enrichment for c-Myb was detected at both c-Myb

binding regions in MDA-MB-231 cells (Fig. 4I), and there was only a slight trend of

suppressed binding of c-Myb to the Axin2 promoter upon β-catenin knockdown

(Supplementary Fig. S4E). Together, these results indicate that c-Myb can directly bind to the

Axin2 promoter to stimulate Axin2 expression and induce an invasive phenotype in MDA-

MB- 231 cells.

Loss of c-Myb suppresses breast cancer metastasis

We next examined the role of c-Myb in metastasis in mice. First, we found that both c-Myb

and Axin2 are up-regulated in a MDA-MB-231-BM (14), a highly metastatic subclone of

MDA-MB-231 that preferentially disseminates to bone (Fig. 5A). We subsequently depleted

endogenous c-Myb expression in MDA-MB-231 BM cells (Supplementary Fig. S5A) and

performed intracardial xenograft experiments in a mouse model for breast cancer metastasis

(32). Mice injected with c-Myb-depleted BM cells developed less bone metastases after

36 days (Fig. 5B). Both BLI signal and number of metastatic nodules were significantly

decreased in the c-Myb-depleted groups (Fig. 5C and D). Importantly, the c-Myb-depleted

cells did not show significant proliferation defects (Supplementary Fig. S5B), in agreement

with previous experiments published for ER- breast cancer cells (4). These results suggest

that c-Myb is critical for breast cancer bone metastasis. We next examined the role of c-Myb

on metastatic dissemination from the primary tumor site to distant organs. For this, we

orthotopically transplanted control 4T1 breast cancer cells or c-myb depleted 4T1 cells into

the mammary fat pad of the BALB/c Mice. Consistent with the positive effects of c-Myb on

mesenchymal and metastatic breast cancer traits, depletion of c-myb blocked slug and axin2

expression in 4T1 cells (Supplementary Fig. S5C and D). At 21 days after orthotopic

implantation, primary tumor formation of 4T1 cells with c-myb knockdown was similar to

that of the control 4T1 cells (Fig 5E). However, the c-myb deficient 4T1 cells displayed a

significant reduction of both number and size of lung metastasis (Fig 5F-H and

Supplementary Fig. S5E). Together, these data show that c-Myb also contributes to breast

cancer metastasis in an orthotopic mouse model.

High c-Myb expression shows a positive correlation with Axin2 expression in clinical

cases

To determine the clinical relevance of the relation between c-Myb and Axin2 shown above,

we performed immunohistochemistry (IHC) staining of tissue microarrays (TMAs) that

included 176 breast cancer samples collected at Leiden University Medical Centre

(Supplementary Table S1; Supplementary Fig. S6A). Nuclear expression of c-Myb and

cytoplasmic expression of Axin2 were subjectively scored as high staining or low staining

(Fig. 6A). As Figure 6B shows, c-Myb up-regulation positively associated with lymph node

invasion. Next, we investigated the correlation between c-Myb and Axin2 in the same tissue

microarray. Consistent with the result that c-Myb activates Axin2 in breast cancer, we

observed that more than 70% of Axin2 high expression samples also have high c-Myb

expression (Fig. 6C). Vice versa the c-Myb levels were significantly positively correlated

with Axin2 expression (Fig. 6D). These data support our findings that c-Myb enhances Axin2

expression and that this induces an aggressive phenotype and poor prognosis in human breast

cancer.

Interleukin-1β (IL-1β) stimulates c-Myb expression in ER- breast cancer

To search for mechanisms that contribute to c-Myb expression in breast cancer, we tested

several cytokines to see if they regulated c-Myb expression in the MDA-MB-231 breast

cancer cell line. The inflammatory cytokine IL-1β was found to increase c-Myb mRNA levels

(Fig. 7A). Similarly, the c-Myb protein level increased significantly from 1h to 10 h after 10

ng/ml IL-1β treatment, peaking at 3 hours (Fig. 7B). We also found a slight trend of increased

Axin2 mRNA expression upon 10h IL-1β treatment, but c-Myb depletion blocked Axin2

induction in both mock or IL-1β treated cells (Supplementary Fig. S6B). Members of the IL-

1 family of cytokines are abundantly expressed in the microenvironment of breast cancer and

are indispensable for tumor progression (33). To test whether c-Myb is functionally involved

in the IL-1β response, we examined the effect of c-Myb depletion on IL-1β-stimulated cell

migration. Interestingly, both basal and IL-1β-induced MDA-MB-231 cell migration was

effectively blocked by c-Myb knockdown (Fig. 7C). Moreover, epidermal growth factor

(EGF) induced migration was also inhibited in the c-Myb deficient MDA-MB-231 cells

(Supplementary Fig. S6C). Axin2 and β-catenin were also required for IL-β induced cell

migration (Supplementary Fig. S6D). These data indicate that one or more (mesenchymal)

target genes of c-Myb and β-catenin are required both for basal migration and for migration

induced by extracellular stimuli. The stimulation of c-Myb by IL-1β prompted us to re-

analyse the TCGA dataset by PROGgene (20,34). For the ER- breast cancer patients, high

expression of both IL-1β and c-Myb mRNA was significantly associated with poor prognosis

(Fig. 7D). Collectively, these results indicate that IL-1β is associated with c-Myb activation

and leads to poor survival in ER- breast cancer.

Discussion

Our study suggests a model in which c-Myb is activated by IL-1β (and other extracellular

cues) and interacts with β-catenin in the nucleus. c-Myb then acts as a co-factor of β-catenin

and accelerates expression of genes that are downstream of β-catenin in breast cancer. In

addition, c-Myb also directly binds to the Axin2 promoter and drives Axin2 expression

enhancing breast tumor progression (Fig. 7E).

c-Myb expression can be induced by ER and high c-Myb predicts good survival in ER+

breast cancers that can be efficiently treated (4,7,35). The role of Myb in EMT, invasion and

metastasis is less clear, and is likely to depend on the exact genetic changes and pathway

defects in the tumor and on the tumor microenvironment. Transcriptional events mediated by

estrogen signaling have a distinct role in development and proliferation in the early stages of

a tumor, and the ER target GATA3 and can negatively regulate metastasis (36), whereas

FOXA1 positively correlates with distant metastasis (37). Moreover, c-Myb can regulate

tumor migration in vitro in both ER+ and ER- cell lines (6,38). Previously, Hugo et al.

concluded that the role of Myb in EMT is most likely context-dependent. These authors

found that the EMT-activator Zeb1 can inhibit c-Myb expression, and that c-Myb and Zeb1

are inversely expressed in PMA42 and MDA-MB-231 cells (39). Hugo et al. postulated that

Myb could drive early EMT by inducing Slug and Snail, but that at later stages Zeb1 could

suppress Myb and also Slug and Snail. It should also be noted in this respect that depending

on the cellular context excess of c-Myb ( e.g. by overexpression) might affect different Myb

target genes and functions than Myb depletion. In the current study, we found that c-Myb is

specifically required for breast cancer invasion and metastasis in experimental xenograft

models in zebrafish and mice. As one of the signature proteins that correlates with ER status,

c-Myb has also been implicated in survival in luminal/ER+ breast cancer and is linked to the

regulation of BCL2 (20,40). However, we observed no significant proliferation or survival

defects after knockdown or ectopic expression of c-Myb/Axin2 in ER- cell lines

(Supplementary Fig. S7). This is in line with previous studies that c-Myb knockdown does

not influence the proliferation of ER- cells (4).

One of the signaling pathways involved in breast cancer progression is the Wnt pathway,

which can induce both EMT and invasion/metastasis (10). Importantly, we found c-Myb to

enhance the expression levels of various target genes of Wnt/β-catenin signaling, including

cyclin D1 and Axin2, by functionally interacting with LEF-1/β-catenin and facilitating β-

catenin binding to its target promoters. In addition, c-Myb was found to bind to the Axin2

promoter directly, and thereby to induce Axin2 expression and enhance breast tumor

progression. High expression of Cyclin D1 promotes local invasion and is associated with

bone metastasis (41). Axin2 can fine-tune Wnt signaling and counteract excessive signaling

as a Wnt feedback inhibitor, but, more importantly, Axin2 also acts as a chaperone for

GSK3β and controls its subcellular location in human breast cancer. As a result of this, Snail

is dephosphorylated and stabilized, contributing to EMT and invasion (13). Moreover, we

previously demonstrated that Axin2 can form a complex with the E3 ligases Rnf12 and

Arkadia and induce Smad7 degradation to promote TGF-β signaling and breast bone

metastasis (42). Importantly, we found a positive correlation between c-Myb and Axin2 in

clinical samples. We further show that c-Myb expression in breast cancer cells can be

activated by IL-1β, which suggest that c-Myb may in particular enhance migration and

invasion in response to inflammatory signals in the tumor microenvironment. In this respect it

is interesting to note that c-Myb can contribute to TGF-β-induced EMT in ER+ breast cancer

cells (6). It might therefore be interesting to investigate the IL-1β/TGF-β/Wnt interplay in this

process.

The exact mechanism by which c-Myb enhances Wnt/β-catenin inducible genes remains to be

established. β-catenin can interact with both LEF1/TCF and Wnt co-activators/repressors

through its ARM domain which consists of 12 repeats (43). Our results demonstrated that c-

Myb binds to the NTAA motif of the ARM domain. Consistent with this model, we observed

decreased expression of Wnt target genes and reduced binding affinity of β-catenin to the

corresponding promoters when c-Myb was knocked down in MDA-MB-231 cells. c-Myb

might therefore act as a β-catenin co-activator. A previous study showed that in colorectal

cancer c-Myb can also cooperate with activated β-catenin via independent binding to the c-

Myc promoter, which highlights the potential interplay between c-Myb and Wnt activation

during cancer development (2).

Our results further suggest that c-Myb positively controls various mesenchymal markers like

Twist, Snail, Slug and N-cadherin to contribute to local invasion and distant metastasis of

cancer. Moreover, Wnt signaling can promote EMT by inducing the expression of these

mesenchymal markers during breast cancer invasion (11), further supporting our observation

that Wnt-β-catenin signaling might contribute to c-Myb-induced cancer progression.

Depletion of c-Myb in MDA-MB-231 cells was found to impair breast cancer bone

metastasis in a mouse model. Therefore, c-Myb appears to be required for the spreading of

advanced metastatic cells. In line with this, we found that silencing of endogenous c-Myb

expression in 4T1 cells has no significant effect on primary formation but suppresses distant

metastasis. Interestingly, a previous report showed that c-Myb overexpression in 4T1 cells

up-regulates the expression of matrix metalloproteinases (MMP) 9 and cathepsin D but

inhibits MMP1, and promotes migration and matrigel invasion, but not collagen invasion. In

addition, c-Myb overexpression in 4T1 cells was found to delay primary tumor growth and to

affect metastasis in an organ-specific manner (38).

Long-term clinical observations and mechanistic studies have shown a strong association of

inflammation with cancer development and progression. Inflammation can result in an influx

of inflammatory cells and secretion of inflammatory cytokines including TNFα, IL-1, IL-6

and IL-8 into the tumor microenvironment, leading to activation of the NF-κB pathway (44).

Clinically, high c-Myb and IL-1β expression were found to be linked with poor survival in

ER- breast cancer. Therefore, our results suggest that levels of c-Myb and IL-1β might be

important to predict the aggressiveness of specific breast cancers.

Author’s contributions

Y.L designed/performed experiments, analysed and interpreted the results and wrote the

manuscript; F. Z and L.Z. designed/ performed experiments and wrote the manuscript; K. J

performed mouse metastasis experiments. G.W.P, and W. E. M performed pathology analysis

of the specimens. X. Y performed online clinical data analysis. H.v. D was involved in

discussing and writing. P.t.D directed the research, interpreted the data and wrote the

manuscript.

Acknowledgments

We thank Dr. Odd Stokke Gabrielsen for the HA-c-Myb plasmid, Dr. Frank Costantini for

Axin2 reporter constructs, Dr. Zhijie Chang for the TOPFlash reporter and all members of ten

Dijke lab for discussion.

Grant Support

This work is supported by the Cancer Genomics Centre Netherlands, Swedish Cancerfonden

(09 0773), Zhejiang Provincial Natural Science Foundation of China (grant number

R14C070002) and The Chinese National Natural Science Funds (grant number 31471315).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

References

1. Biroccio A, Benassi B, D'Agnano I, D'Angelo C, Buglioni S, Mottolese M, et al. c-Myb and Bcl-x overexpression predicts poor prognosis in colorectal cancer: clinical and experimental findings. The American journal of pathology 2001;158(4):1289-99. 2. Ciznadija D, Tothill R, Waterman ML, Zhao L, Huynh D, Yu RM, et al. Intestinal adenoma formation and MYC activation are regulated by cooperation between MYB and Wnt signaling. Cell death and differentiation 2009;16(11):1530-8. 3. Kauraniemi P, Hedenfalk I, Persson K, Duggan DJ, Tanner M, Johannsson O, et al. MYB oncogene amplification in hereditary BRCA1 breast cancer. Cancer research 2000;60(19):5323-8. 4. Drabsch Y, Hugo H, Zhang R, Dowhan DH, Miao YR, Gewirtz AM, et al. Mechanism of and requirement for estrogen-regulated MYB expression in estrogen-receptor-positive breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America 2007;104(34):13762-7. 5. Miao RY, Drabsch Y, Cross RS, Cheasley D, Carpinteri S, Pereira L, et al. MYB is essential for mammary tumorigenesis. Cancer research 2011;71(22):7029-37. 6. Cesi V, Casciati A, Sesti F, Tanno B, Calabretta B, Raschella G. TGFbeta-induced c-Myb affects the expression of EMT-associated genes and promotes invasion of ER+ breast cancer cells. Cell cycle 2011;10(23):4149-61. 7. Ramsay R. Myb: getting involved in EMT. Cell cycle 2012;11(3):433-4. 8. Janda CY, Waghray D, Levin AM, Thomas C, Garcia KC. Structural basis of Wnt recognition by Frizzled. Science 2012;337(6090):59-64. 9. Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell 2012;149(6):1192-205. 10. Dey N, Barwick BG, Moreno CS, Ordanic-Kodani M, Chen Z, Oprea-Ilies G, et al. Wnt signaling in triple negative breast cancer is associated with metastasis. BMC cancer 2013;13(1):537. 11. DiMeo TA, Anderson K, Phadke P, Fan C, Perou CM, Naber S, et al. A novel lung metastasis signature links Wnt signaling with cancer cell self-renewal and epithelial-mesenchymal transition in basal-like breast cancer. Cancer research 2009;69(13):5364-73. 12. Lin SY, Xia W, Wang JC, Kwong KY, Spohn B, Wen Y, et al. Beta-catenin, a novel prognostic marker for breast cancer: its roles in cyclin D1 expression and cancer progression. Proceedings of the National Academy of Sciences of the United States of America 2000;97(8):4262-6. 13. Yook JI, Li XY, Ota I, Hu C, Kim HS, Kim NH, et al. A Wnt-Axin2-GSK3beta cascade regulates Snail1 activity in breast cancer cells. Nature cell biology 2006;8(12):1398-406. 14. Deckers M, van Dinther M, Buijs J, Que I, Lowik C, van der Pluijm G, et al. The tumor suppressor Smad4 is required for transforming growth factor beta-induced epithelial to

mesenchymal transition and bone metastasis of breast cancer cells. Cancer research 2006;66(4):2202-9. 15. Li Y, Drabsch Y, Pujuguet P, Ren J, van Laar T, Zhang L, et al. Genetic depletion and pharmacological targeting of alphav integrin in breast cancer cells impairs metastasis in zebrafish and mouse xenograft models. Breast cancer research : BCR 2015;17:28. 16. Zhang X, Zhang J, Zhang L, van Dam H, ten Dijke P. UBE2O negatively regulates TRAF6- mediated NF-kappaB activation by inhibiting TRAF6 polyubiquitination. Cell research 2013;23(3):366-77. 17. Drabsch Y, He S, Zhang L, Snaar-Jagalska BE, Ten Dijke P. Transforming growth factor-beta signalling controls human breast cancer metastasis in a zebrafish xenograft model. Breast cancer research : BCR 2013;15(6):R106. 18. Tai LH, Tanese de Souza C, Sahi S, Zhang J, Alkayyal AA, Ananth AA, et al. A mouse tumor model of surgical stress to explore the mechanisms of postoperative immunosuppression and evaluate novel perioperative immunotherapies. Journal of visualized experiments : JoVE 2014(85). 19. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Science signaling 2013;6(269):pl1. 20. Cancer Genome Atlas N. Comprehensive molecular portraits of human breast tumours. Nature 2012;490(7418):61-70. 21. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 2012;486(7403):346-52. 22. Radvanyi L, Singh-Sandhu D, Gallichan S, Lovitt C, Pedyczak A, Mallo G, et al. The gene associated with trichorhinophalangeal syndrome in humans is overexpressed in breast cancer. Proceedings of the National Academy of Sciences of the United States of America 2005;102(31):11005-10. 23. Zhao H, Langerod A, Ji Y, Nowels KW, Nesland JM, Tibshirani R, et al. Different gene expression patterns in invasive lobular and ductal carcinomas of the breast. Molecular biology of the cell 2004;15(6):2523-36. 24. Gonda TJ, Leo P, Ramsay RG. Estrogen and MYB in breast cancer: potential for new therapies. Expert opinion on biological therapy 2008;8(6):713-7. 25. Yilmaz M, Christofori G. EMT, the cytoskeleton, and cancer cell invasion. Cancer metastasis reviews 2009;28(1-2):15-33. 26. Sotiriou C, Wirapati P, Loi S, Harris A, Fox S, Smeds J, et al. Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis. Journal of the National Cancer Institute 2006;98(4):262-72.

27. Nguyen DX, Chiang AC, Zhang XH, Kim JY, Kris MG, Ladanyi M, et al. WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis. Cell 2009;138(1):51-62. 28. Zhou F, Zhang L, van Laar T, van Dam H, Ten Dijke P. GSK3beta inactivation induces apoptosis of leukemia cells by repressing the function of c-Myb. Molecular biology of the cell 2011;22(18):3533-40. 29. Stadeli R, Hoffmans R, Basler K. Transcription under the control of nuclear Arm/beta-catenin. Current biology : CB 2006;16(10):R378-85. 30. Jho EH, Zhang T, Domon C, Joo CK, Freund JN, Costantini F. Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Molecular and cellular biology 2002;22(4):1172-83. 31. Andersson KB, Berge T, Matre V, Gabrielsen OS. Sequence selectivity of c-Myb in vivo. Resolution of a DNA target specificity paradox. The Journal of biological chemistry 1999;274(31):21986-94. 32. Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM, Cordon-Cardo C, et al. A multigenic program mediating breast cancer metastasis to bone. Cancer cell 2003;3(6):537-49. 33. Pantschenko AG, Pushkar I, Anderson KH, Wang Y, Miller LJ, Kurtzman SH, et al. The interleukin-1 family of cytokines and receptors in human breast cancer: implications for tumor progression. International journal of oncology 2003;23(2):269-84. 34. Goswami CP, Nakshatri H. PROGgene: gene expression based survival analysis web application for multiple cancers. Journal of clinical bioinformatics 2013;3(1):22. 35. Thorner AR, Parker JS, Hoadley KA, Perou CM. Potential tumor suppressor role for the c-Myb oncogene in luminal breast cancer. PloS one 2010;5(10):e13073. 36. Yan W, Cao QJ, Arenas RB, Bentley B, Shao R. GATA3 inhibits breast cancer metastasis through the reversal of epithelial-mesenchymal transition. The Journal of biological chemistry 2010;285(18):14042-51. 37. Ross-Innes CS, Stark R, Teschendorff AE, Holmes KA, Ali HR, Dunning MJ, et al. Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature 2012;481(7381):389-93. 38. Knopfova L, Benes P, Pekarcikova L, Hermanova M, Masarik M, Pernicova Z, et al. c-Myb regulates matrix metalloproteinases 1/9, and cathepsin D: implications for matrix- dependent breast cancer cell invasion and metastasis. Molecular cancer 2012;11:15. 39. Hugo HJ, Pereira L, Suryadinata R, Drabsch Y, Gonda TJ, Gunasinghe NP, et al. Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells. Breast cancer research : BCR 2013;15(6):R113. 40. Drabsch Y, Ramsay RG, Gonda TJ. MYB suppresses differentiation and apoptosis of human breast cancer cells. Breast cancer research : BCR 2010;12(4):R55.

41. Dai M, Al-Odaini AA, Fils-Aime N, Villatoro MA, Guo J, Arakelian A, et al. Cyclin D1 cooperates with p21 to regulate TGFbeta-mediated breast cancer cell migration and tumor local invasion. Breast cancer research : BCR 2013;15(3):R49. 42. Zhou F, Drabsch Y, Dekker TJ, de Vinuesa AG, Li Y, Hawinkels LJ, et al. Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-beta signalling. Nature communications 2014;5:3388. 43. Huber AH, Nelson WJ, Weis WI. Three-dimensional structure of the armadillo repeat region of beta-catenin. Cell 1997;90(5):871-82. 44. Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature 2008;454(7203):436-44.

Figure Legends

Figure 1. c-Myb is a potential activator of breast cancer invasion. A, Oncomine plots of c-

Myb mRNA levels in normal breast tissue (normal), invasive mixed breast carcinoma

(IMBC), invasive ductal breast carcinoma (IDBC), and invasive lobular breast carcinoma

(ILBC) in public breast cancer datasets (20-23). B, Real-time quantitative PCR (left) or

immunoblotting analysis (right) of cells stably expressing control shRNA (con sh) or two

independent c-Myb shRNAs (sh1 and 2). Mean ± s.d. of triplicates, **p<0.01 ***p<0.001. C,

Transwell migration of control or c-Myb depleted cells. Upper: representative images of

migrated cells. Lower: fold changes of migrated cell numbers, mean ± s.d. of triplicates. D,

qPCR analysis in control or c-Myb depleted (sh 1 and 2) cells as indicated. Mean ± s.d. of

triplicates, *p<0.05 **p<0.01 ***p<0.001. E, Immunoblotting of mesenchymal markers

expression as indicated. F, Correlation between c-Myb mRNA and N-Cadherin, Snail, Slug

mRNA expression in a clinical breast cancer dataset (GSE2990) (26). Scale: log2 median-

centred value of gene expression. Pearsons’ coefficient tests were conducted to calculate

statistical significance. G, Relapse free survival analysis of patients with breast cancer with

low or high c-Myb mRNA expression in the same breast cancer dataset.

Figure 2. c-Myb positively controls breast cancer progression in a zebrafish model. A,

Schematic representation of the zebrafish embryo xenograft assay. B, Representative

overview images at 6 days post implantation (dpi) of zebrafish injected with MDA-MB-231

cells. Arrows: tumor cells, scale bar: 500 μm. C, Detailed images of the tail fin. Arrows:

tumor cells, scale bar: 100 μm. D, Area of metastatic cells in individual zebrafish embryos

(n=29). **p<0.01 ***p<0.001. E, Percentage of the zebrafish embryos displaying tumor

progression to the tail fin . ***p<0.001. F, Immunoblotting of c-Myb in control or c-Myb

overexpressing MCF10A-M2 cells. G, Overview image at 6 dpi of zebrafish embryos

implanted with MCF10A-M2 cells. Arrows: tumor cells, scale bar: 500 μm. H, Detailed

images of the tail fin. Arrows: tumor cells, scale bar: 100 μm. I, Area of metastatic cells in

individual zebrafish embryos (n=33). *p<0.05. J, Percentage of the zebrafish embryos

displaying positive tumor progression to the tail fin. **p<0.01.

Figure 3. c-Myb interacts with β-catenin and affects Wnt/β-catenin target gene expression. A,

Correlation heatmap of c-Myb and Wnt target gene expression. Rows and columns are ranked

by pearson correlation. Red: high correlation; green: low correlation. Branches represent

relations between each gene profile of 534 breast patients in TCGA dataset (20). *** p<0.001.

B, Ectopic expression of c-Myb potentiates TOPFlash (but not FOPFlash) reporter activity in

HEK293T cells with or without 100ng/ml recombinant Wnt3a treatment in a dose dependant

manner. *p<0.05, ** p<0.01. C, Effect of c-Myb depletion on the expression of Axin2 and

Cyclin D1 in MDA-MB-231 and MCF10A-M2 cells upon recombinant Wnt3 treatment

(100ng/ml). Mean ± s.d. of triplicates. *p<0.05, ** p<0.01. D, Co-immunoprecipitation (Co-

IP) of HA-c-Myb and Flag-β-catenin transfected in HEK293T cells as indicated (IP:

immunoprecipitation; IB: immunoblot). E, Endogenous interaction between c-Myb and β-

catenin detected by immunoblotting of Co-IP samples from MDA-MB-231 and MCF10-M2

cells. F, Upper: schematic representation of HA-β-catenin deletion mutants tested. Lower:

Co-IP of Myc-c-Myb and HA-β-catenin deletion mutants transfected in HEK293T cells and

detected by immunoblotting. G, Endogenous interaction between LEF-1 and β-catenin in

control and c-Myb knockdown MDA-MB-231 cells detected by immunoblotting of Co-IP

samples. H, Chromatin immunoprecipitation (ChIP) of β-catenin in control and c-Myb

depleted MDA-MB-231 cells. Upper: Sequence and location of the analysed TCF binding

motifs in the Axin2 (48 from transcription start site) and Cyclin D1 (-23 from transcription

start site) genes. Lower: chromatin-bound β-catenin detected by qRT-PCR. The Actin

promoter served as control promoter (con pro). Input genomic DNA was used for

normalization, mean ± s.d. of triplicates.

Figure 4.c-Myb-induced breast cancer metastasis in zebrafish mediated via Axin2 induction.

A, Axin2 protein levels in control and c-Myb depleted MDA-MB-231 cells as detected by

immunoblotting. B, Immunoblot detection of the c-Myb and Axin2 protein levels in MDA-

MB-231 cells infected with lentivirus vectors as indicated. C, Representative overview

images at 6 days post implantation (dpi) of zebrafish injected with MDA-MB-231 cells

shown under B. Arrows: tumor cells, scale bar: 500 μm. D, Detailed images of the tail fin.

Arrows: tumor cells, scale bar: 100 μm. E, Area of metastatic cells in individual zebrafish

embryos (n = 30). F, Percentage of the zebrafish embryos displaying tumor progression to the

tail fin. con sh vs Axin2 sh, c-Myb, or c-Myb oe Axin2 sh; ***p<0.001. G, Ectopic

expression of c-Myb induces an Axin2 promoter-driven luciferase reporter in HEK293T cells.

**p<0.01 ***p<0.001. H, Schematic diagram of the Axin2 promoter showing three c-Myb

binding sites and the 2 binding regions (red) analysed by chromatin immunoprecipitation in

(I). I, Chromatin IP (ChIP) of Axin2 promoter-bound c-Myb in MDA-MB-231 cells. qPCR

was performed with primers for the 2 c-Myb binding regions shown in (G). The Actin

promoter served as control promoter (con pro). Input genomic DNA was used for

normalization, mean ± s.d. of triplicates. **p<0.01

Figure 5. c-Myb knockdown in MDA-MB-231 BM cells impairs bone metastasis in mice. A,

c-Myb and Axin2 protein in MDA-MB-231 cells from ATCC and the BM subline detected

by immunoblotting. B, Representative bioluminescent (BLI) images of mice injected

intracardially with control or c-Myb depleted MDA-MB-231 BM cells at 36 days after

injection. C, Normalized BLI signals in mice injected with control or c-Myb depleted MDA-

MB-231 BM cells at 28 and 36 days after injection. D, Number of bone metastasis nodules at

36 days after injection. The nodules were analysed by detected BLI signal in the bones in

each mouse in each experimental group. E, Primary tumor formation in mice after orthotopic

injection of control or c-Myb depleted 4T1 cells at day 21 after implantation. Left,

representative BLI signals of primary tumor. Right, normalized BLI signals. F,

Representative BLI signals of lung metastasis at 21 days after injection. G, Number of lung

metastasis nodules. H, Normalized BLI signals of isolated lung.

Figure 6. c-Myb correlates with Axin2 in breast cancer patients. A, Representative images of

c-Myb and Axin2 immunohistochemistry of breast cancer tissue. Upper: objective: 5×, scale

bar: 200µm. Lower: objective: 25×. B, Percentage of patient samples with high or low c-Myb

expression showing in negative (LN-) or positive (LN+) lymph node invasion. C, Percentage

of samples with low or high c-Myb expression in relation to the expression levels of Axin2.

D, Correlation analysis of c-Myb and Axin2 expression in the same tissue microarray. χ2 =

12.136, p = 0.0001.

Figure 7. c-Myb is regulated by IL-1β. A, qPCR analysis of c-Myb mRNA levels in MDA-

MB-231 cells treated with 10 ng/ml IL-1β in the presence of 1% serum for the indicated

times. *p<0.05, **p<0.01. B, Left, immunoblotting of c-Myb protein in MDA-MB-231 cells

treated with IL-1β in the presence of 1% serum for the indicated times. Right, quantification

of the protein levels. Mean ± s.d. , *p<0.05 **p<0.01 ***p<0.001. C, Transwell migration of

control or c-Myb depleted MDA-MB-231 cells treated with or without 10ng/ml IL-1β in the

presence of 1% serum for 16 h. Left, representative images of migrated cells. Right, fold

changes of migrated cell numbers, mean ± s.d. of triplicates. D, Overall survival analysis of

patients in breast cancer with both low c-Myb and IL-1β or both high c-Myb and IL-1β

mRNA expression in TCGA ER- breast cancer dataset by PROGgene. E, Proposed model of

the role of c-Myb in breast cancer.

c-Myb enhances breast cancer invasion and metastasis through the Wnt/ β-catenin/Axin2 pathway

Yihao Li, Ke Jin, Gabi W van Pelt, et al.

Cancer Res Published OnlineFirst April 5, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-2302

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