Full text

Turn on search term navigation

ARTICLE

Received 23 Mar 2013 | Accepted 12 Jul 2013 | Published 12 Aug 2013

NF-kB activation, induced by inammatory cytokines or by epigenetically dysregulated NIK expression, cell-autonomously upregulates JAG1 expression in non-cancer stem cells. This upregulation stimulates NOTCH signalling in cancer stem cells in trans, leading to an expansion of cancer stem cell populations. Among breast cancers, the NF-kB-dependent induction of JAG1 and the NOTCH-dependent expansion of the cancer stem cell population occur only in the basal-like subtype. Collectively, our results indicate that NF-kB has a non-cell-autonomous role in regulating cancer stem cell populations by forming intratumoural microenvironments composed of JAG1-expressing non-cancer stem cells with a basal-like subtype.

DOI: 10.1038/ncomms3299

NF-kB non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype

Mizuki Yamamoto1,2, Yuu Taguchi1, Taku Ito-Kureha3, Kentaro Semba2, Noritaka Yamaguchi4 & Jun-ichiro Inoue1

1 Division of Cellular and Molecular Biology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan. 2 Department of Life Science and Medical Bio-Science, Waseda University, Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan. 3 Cell Signal Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, Kunigami-gun, Okinawa 904-0495, Japan. 4 Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan. Correspondence and requests for materials should be addressed to J.-i.I. (email: mailto:[email protected]

Web End [email protected] ).

NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 1

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299

Gene expression studies have established ve breast cancer subtypes: luminal-like, v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2)-enriched,

basal-like, claudin-low and normal-breast-like13. The luminal-like subtype is characterized as displaying an oestrogen-receptor-positive (ER ) and progesterone-receptor-positive (PR )

phenotype, whereas the basal-like and claudin-low subtypes constitute the majority of triple-negative breast cancers (TNBCs; that is, ER , PR and ERBB2 ). Although selective oestrogen-receptor modulators, aromatase inhibitors and trastuzumab are effective drugs for treating the luminal-like and ERBB2-enriched subtypes, TNBC displays high malignancy and exhibits a poor prognosis in response to various therapies4. To elucidate the signalling pathways that contribute to the malignant TNBC phenotype, we investigated NF-kB because NF-kB activation in

TNBC is signicantly higher than in other subtypes5.

The NF-kB family consists of ve members, namely, p50, p52, RelA, RelB and c-Rel, which form homo- and heterodimers to activate the transcription of target genes responsible for inammation, immunoregulation and cell differentiation6. When forming complexes with members of the IkB family, including IkBa, IkBb, IkBe, p105 (a precursor to p50) and p100 (a precursor to p52), NF-kB is transcriptionally inactivated due to its cytoplasmic sequestration. In the canonical pathway, many stimuli, including various cytokines and bacterial and viral products, induce the IkB kinase b (IKKb)-catalysed phosphorylation of IkBa and its proteasomal degradation, which is followed by the nuclear translocation of p50-RelA heterodimers7. The non-canonical pathway is activated by receptors that are required for the formation of lymphoid organs and lymphocyte development, such as the lymphotoxin-b receptor, the receptor activator of NF-kB (RANK) and CD40.

This pathway induces the IKKa-catalysed phosphorylation of the C-terminal half of p100, which sequesters RelB to the cytoplasm. This process leads to the polyubiquitination-dependent processing of p100 to p52, which is followed by the nuclear translocation of the p52-RelB heterodimers8. Most importantly, because of various negative regulators, activation of the two pathways is largely transient, enabling homoeostasis in inammatory or immune responses9. Nevertheless, TNBC undergoes constitutive, strong NF-kB activation. Furthermore, the adenovirus-mediated expression of the non-degradable IkBa super-repressor (IkBaSR), in which Ser-32 and -36 (the residues phosphorylated by IKKb) were substituted with alanine, blocked NF-kB activation and thereby inhibited the growth of several TNBC cell lines5. This result suggests that constitutive NF-kB activation has a critical role in TNBC malignancy.

Cancer stem cells (CSCs), a subpopulation within a tumour, can self-renew and differentiate into various tumour cell types, thereby reconstituting intratumoural heterogeneity10. Moreover, CSCs are highly tumorigenic, relatively resistant to conventional chemotherapy and radiotherapy and involved in both tumour initiation and metastasis11. Therefore, the role of NF-kB activation in regulating CSC populations in TNBC is of particular interest. Although most previous studies have proposed that NF-kB activation in CSCs cell-autonomously regulates the CSC population, the molecular mechanisms by which NF-kB regulates CSCs are not fully understood1215.

Furthermore, because each breast cancer subtype is derived from mammary epithelial cells at a distinct differentiation stage, the molecular mechanisms for regulating CSC population are likely to differ among subtypes1619.

In this study, we sought to determine the role of NF-kB in regulating the CSC population in the basal-like breast cancer subtype and identied a subtype-specic, NF-kB-JAG1 axis that regulates CSC population size.

ResultsNF-kB regulates CSC population in the basal-like subtype. To analyse the relationship between NF-kB and the CSC population, we rst assessed the correlation between NF-kB activation and the CSC population size, dened as the CD24

CD44highEpCAM fraction20 (Fig. 1a) or the ALDEFLOUR fraction (Fig. 1b) (refs 21,22). Interestingly, the CSC population sizes in the basal-like cells were clearly correlated with the NF-kB activation levels in both CSC denitions (Fig. 1a,b). In contrast, a correlation between NF-kB activation and the size of the

ALDEFLOUR CSC fractions was scarcely observed in either the luminal-like or the claudin-low cells, although all of the claudinlow cell lines showed high-NF-kB activation levels (Fig. 1b, the

CD24 CD44highEpCAM fractions were not recognized in the luminal-like and claudin-low subtypes, as shown in Fig. 1a).

These results led us to hypothesize that NF-kB may regulate CSC populations, specically in basal-like cells. To conrm this hypothesis, we generated both low-NF-kB cells, in which

IkBaSR was expressed to block NF-kB activation, and high-NF-kB cells, in which the constitutively active form of IKKb (IKKbEE, generated by converting Ser-176 and Ser-180 in the activation loop into glutamic acid) or wild-type IKKb was expressed to enhance NF-kB activation (Fig. 1c). The low-NF-kB cells showed a reduced CSC population, whereas the high-NF-kB cells showed an expanded CSC population in basal-like cells (Fig. 1d, Supplementary Fig. S1). The modulation of NF-kB activation did not affect cell growth or survival (Supplementary Fig. S2). However, this NF-kB-dependent regulation of the CSC population was not observed in the claudin-low or luminal-like cells (Fig. 1e,f). Under conditions in which approximately 90% of the mammospheres were derived from a single cell (Supplementary Fig. S3), the low-NF-kB cells showed reduced mammosphere formation, whereas the high-NF-kB cells showed enhanced formation in basal-like cells (Fig. 1g). However, this NF-kB-dependent regulation of sphere-formation ability was not observed in the claudin-low or luminal-like cells (Fig. 1h). Tumour necrosis factor-a (TNFa) is a well-known cytokine that induces NF-kB activation and is involved in tumour malignancy23. TNFa stimulation, which induced RELB expression (indicating NF-kB activation), signicantly expanded the CSC population (Fig. 1i) and enhanced the mammosphere-forming ability (Fig. 1j) of the basal-like cells. To further conrm the NF-kB-induced CSC population expansion, we analysed the effects of

NF-kB activation on tumorigenicity. HCC1937 cells and cells containing modulated NF-kB activation were injected with Matrigel into the mammary fat pads of NOD/SCID mice, and palpable tumours were counted 10 weeks after injection. The low-NF-kB cells showed signicantly lower tumorigenicity than the control cells, and the high-NF-kB cells displayed substantially higher tumorigenicity.

NF-kB in non-CSCs is crucial in regulating CSC population. To elucidate the molecular mechanisms of the NF-kB-mediated regulation of the CSC population, we rst compared the NF-kB activities in the CSC and non-CSC fractions. Each cell fraction was isolated from two basal-like cells, MB468 and HCC1937 (Fig. 2a). Electrophoretic mobility shift assays (EMSAs) revealed that the NF-kB activities in the CSC and non-CSC fractions were indistinguishable in both cell lines (Fig. 2b), leading us to hypothesize that NF-kB activation in non-CSCs may regulate the

CSC population. This hypothesis is in contrast to a previously proposed model, in which NF-kB activation in CSCs cell-autonomously maintains stemness1215. To test this hypothesis, three basal-like cell lines with intact NF-kB activation were labelled with green uorescent protein (GFP) and then co-cultured with a

2 NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299 ARTICLE

Luminal-like

T47D MCF7 MB453

Basal-like

HCC1937 HCC1937

MB231 BT549 MB436

Claudin-low Luminal-like Basal-like Claudin-low

MB231 BT549 MB436

T47D MCF7 MB453

Virus:

Cont

IKK

Cont

IKK

CD24 /CD44high /EpCAM+

CSC population (%)

HCC1143 HCC1143

Basal-like:

ALDEFLUOR+

CSC population (%)

HCC1937

BT549

HCC70

HCC38 HCC38

MB468

Claudin-low:

MB231

Luminal-like:

HCC1954

HCC1954 HCC1954

MB468 MB468

T47D

MCF7

0 10 20 30 40 50 Relative NF-B activation

HCC1143

Basal-like

HCC1954

Claudin-low Luminal-like

MB468

CD24 /CD44high /EpCAM+

CSC population (%)

CD24 /CD44high /EpCAM+

CSC population (%)

HCC1143

BT549 T47D MCF7

MB231

0 0

HCC1937

0.8

NS NS

** **

ALDEFLUOR+

CSC population (%)

15 1.5

0.5

25 *

0.4

0Virus: Virus:

0 0

Cont

IKK

Cont

IKK

Cont

IKK

Cont

IKK

Cont

IKK

Cont

IKK

Cont

IKK

Cont

IKK

HCC1937 MB231

MCF7

HCC1937

HCC1954

MB231 MCF7

NS NS

*** ***

Virus:

Cont

IKK

Sphere formation (%)

Virus:

Cont

HCC1954

Sphere formation (%)

***

IKK

Virus:

Cont

IKK

Virus:

Cont

IKK

HCC1937

TNF: +

MB468 HCC1954

HCC1937

Cont

5/10

3/12

IKK (tumours/injections)

** *

0.5

***

* *

Sphere formation (%)

100

Tumour induction (%)

10/10 10/10 10/10

4/10

0 + +

50 (kDa)

RELB

Tubulin

50 (kDa)

Cont

TNF

50 (kDa)

1,000 10,000 (cells)

Figure 1 | NF-kB regulates the CSC population in basal-like breast cancer cell lines. (a) The correlation between constitutive NF-kB activationand the CD24 /CD44high/EpCAM CSC population in various breast cancer cell lines. A linear regression analysis of values derived from the basal-like cell lines is shown as a dotted line (n 3, means.d., P 0.011; Pearsons correlation coefcient). Relative NF-kB activities were determined as described

in Methods. (b) The correlation between constitutive NF-kB activation and the ALDEFLUOR CSC population in various breast cancer cell lines.

A linear regression analysis of values derived from the basal-like cell lines is shown as a dotted line (n 3, means.d., P 0.015; Pearsons correlation

coefcient). (c) EMSA of NF-kB activation in control, IkBaSR and IKKb-expressing breast cancer cells. (d) The percentages of the CD24 /CD44high/ EpCAM CSC populations (n 3, means.d.; Students t-test) in basal-like cell lines with various NF-kB activation levels. (e,f) The percentages of the

ALDEFLUOR CSC populations (n 3, means.d.; Students t-test) in the claudin-low (e) and luminal-like (f) cell lines with various levels of NF-kB

activation. (g,h) NF-kB regulates the sphere-forming ability of basal-like cells (g), but not that of claudin-low and luminal-like cell lines (h); images showing sphere formation. Scale bar, 500 mm. (i) Fluorescence-activated cell sorting analysis of the CSC population in basal-like cell lines with or without

TNFa treatment (10 ng ml 1) for 3 days. The percentages of the CSC populations (n 3, means.d.; Students t-test) and the expression of RELB, an

indicator of NF-kB activation, with or without TNFa for 24 h. (j) Cells were cultured with or without 10 ng ml 1 TNFa for 3 days and analysed using the sphere-formation assay (n 3, means.d.; Students t-test). (k) The indicated numbers of cells were transplanted into the mammary fat pads of

NOD/SCID mice. For 10 weeks, the mice were examined weekly for tumours by palpation. Statistically signicant differences in tumorigenicity between the control and NF-kB-modied cells were determined using Fishers exact test. All data are representative of three independent experiments. NS, not signicant. *Po0.05, **Po0.01 and ***Po0.001 (dj).

NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 3

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299

CD24high

(non-CSC)

CD24

(CSC)

CD24high

(non-CSC)

GFP+

cells

99.4%

Non-CSC

Sphere formation (%)

15 ** ***

Without co-culture

MB468

0.76% 33.7%

65.8%

99.5% HCC1937

CSC

95.6%

Non-CSC

CSC

91.7%

0.2 %

NF-B-modified cells (Cont, lB- or IKK-expressing)

0.45 %

NF-B

CD24

Cont

IKK

8.3 %

CD24

(CSC)

Sortedcells after co-cultured with cells expressing

GFP+

Oct-1

24.6%

74.88 %

CSC population (Fig. 2d) Sphere formation (Fig. 2e) NF-B activation (Fig. 2f)

CD44

MB468

HCC1937

CD24/CD44high/EpCAM+

CSC population in GFP+ cells (%)

CD24/CD44high/EpCAM+

CSC population in GFP+ cells (%)

*** **

Cont

IKK

GFP+

***

25 HCC1937 2.0 0.8

MB468 HCC1954

Sorted Sorted NF-B-modified cells expressing

cells after co-cultured with cells expressing

0 0 3 6

Days

9 12

** **

1.5

1.0

0.5

0 3 6

Days

9 12

0.6

0.4

** 0.2

0 3 6 9 12 Days

** **

Cont

IKK

+ Co-culture

Analysis of cells

Cont

GFP+

IKK

* *

** NF-B

Oct1

Top

Cont IKK

+IKK

0 Cont

GFP+ GFP+ GFP+

Bottom +Cont +Cont

Cont Cont

TNF

IKK NS

CD24/CD44high/EpCAM+

CSC population (%)

CD24/CD44high/EpCAM+

CSC population (%)

***

40 *** NS

None

CD24/CD44high/EpCAM+

CSC population (%)

NS NS **

Sphere formation (%)

60 ** NS NS

NS NS

Sphere formation (%)

NS NS

Cont

TNF

IL6

IL8

Cont

None

Cont lgG

Anti-IL6

Anti-IL8

Anti-TNF

Cont lgG

Anti-IL6

Anti-IL8

Anti-TNF

None

Cont lgG

Anti-IL6

Anti-IL8

Anti-TNF

Figure 2 | Non-cell-autonomous role of NF-kB in the regulation of CSC populations of basal-like cell lines. (a) Characterization of CSC and non-CSC fractions. CSC and non-CSC fractions of MB468 and HCC1937 cells were isolated according to their CD24, CD44 and EpCAM expression levels.

(b) Nuclear extracts were prepared from the sorted CSC and non-CSC populations presented in a and subjected to EMSAs. Arrows indicate the specic NF-kB/DNA complexes. (cf) NF-kB regulates the CSC population in trans. Schematic representation of the protocol for the co-culture assay (c).

GFP-labelled cells were co-cultured with a 10-fold excess of cells with modied NF-kB activation. Following the indicated number of days of co-culture, the CSC populations of the GFP-labelled cells (n 3, means.d.; Students t-test) were measured (d). After 12 days of co-culture, the GFP-labelled cells were

sorted and evaluated for sphere-forming ability (n 3, means.d.; Students t-test) (e); nuclear extracts were then prepared from the GFP-labelled

and unlabelled cells for the EMSAs (f). (g) Contact between the GFP-labelled and IKKb-expressing cells was required for the efcient expansion of the CSC population of GFP-labelled cells. GFP-labelled cells in the bottom chamber were co-cultured with control cells alone or with IKKb-expressing cells in the same or the top chamber. After 3 days of co-culture, the CSC populations of GFP-labelled cells (n 3, means.d.; Students t-test) in the bottom chamber

were determined. (h) HCC1937 cells were untreated or treated with IL-6 (50 ng ml 1), IL-8 (100 ng ml 1) or TNFa (10 ng ml 1) for 3 days and then subjected to uorescence-activated cell sorting (FACS) analysis and sphere-formation assay (n 3, means.d.; Students t-test). (i) HCC1937 cells were

treated with TNFa (10 ng ml 1) for 3 days in the presence of various neutralizing antibodies (2 mg ml 1) and then subjected to FACS analysis and sphere-formation assay (n 3, means.d.; Students t-test). (j) Control or IKKb-expressing HCC1937 cells were treated with various neutralizing antibodies

(2 mg ml 1) for 3 days and subjected to FACS analysis and sphere-formation assay (n 3, means.d.; Students t-test). All data are representative of three

independent experiments. NS, not signicant. *Po0.05, **Po0.01 and ***Po0.001 (d,e,gj).

10-fold excess of an unlabelled corresponding cell line that was engineered to confer modulated NF-kB activation (Fig. 2c).

Following the co-culture, the populations of GFP CSCs among all of the GFP cells were measured. The co-culture containing the high-NF-kB cells displayed marked expansion of the GFP

CSC population, whereas a signicant reduction in the GFP CSC population was observed in the co-culture containing the low-NF-kB cells (Fig. 2d). Moreover, compared with GFP cells sorted after co-culture with cells displaying intact NF-kB activation, the GFP cells sorted after co-culture with high-NF-kB

4 NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299 ARTICLE

cells or low-NF-kB cells showed higher and lower mammo-sphere-formation activities, respectively (Fig. 2e). Based on the EMSAs of the GFP and GFP cells isolated after the co-culture, the NF-kB activation levels of the GFP cells were not signicantly affected by their co-culture, with the GFP cells displaying different levels of NF-kB activation (Fig. 2f). This result indicates that the level of NF-kB activation in CSCs is not critical for the regulation of the CSC population. Furthermore, the result demonstrates that the non-labelled high- or low-NF-kB cells (the vast majority of which are non-CSCs) stimulate neighbouring CSCs in trans to regulate the CSC population without affecting CSC NF-kB activation levels. These ndings reveal a non-cell-autonomous role for NF-kB in the regulation of the CSC population. The trans-stimulation by non-CSCs was signicantly reduced when the GFP cells were cultured separately from the high-NF-kB cells in trans-well chambers (Fig. 2g), indicating the requirement for direct cellcell contact between the GFP cells and the high-NF-kB cells to promote the efcient regulation of the CSC population. Nevertheless, because partial

CSC population expansion was observed in the absence of cell cell contact (Fig. 2g), we tested the involvement of IL-6 and IL-8, soluble cytokines that are known to be induced by NF-kB and regulate the CSC population22,24, in this partial effect. Neither IL-6 nor IL-8 induced the expansion of the CSC population and enhanced sphere formation in the basal-like cell lines (Fig. 2h, Supplementary Fig. S4). Neutralizing antibodies against IL-6 and IL-8 could not inhibit the TNFa-induced or IKKb-mediated expansion of the CSC population and enhancement of sphere formation (Fig. 2i,j, Supplementary Fig. S4). These results indicate that the cellcell contact-independent CSC expansion is not mediated by IL-6 or IL-8.

JAG1 is an NF-kB-inducible NOTCH ligand. To identify molecules that mediate the cellcell interactions involved in NF-kB-dependent CSC regulation, we focused on the NOTCH signal because NOTCH reportedly transduces signals that maintain the CSC population in breast cancer2527. Expression of the NOTCH target genes HEY1 and HEY2 in CSCs was higher than that in non-CSCs (Fig. 3a). Moreover, HEY1 and HEY2 expression in CSCs was markedly enhanced by elevated NF-kB activation, but was not signicantly affected in non-CSCs (Fig. 3a). These results suggest that the NOTCH signalling in CSCs is more highly activated than that in non-CSCs and that NF-kB activation further enhances NOTCH activation in CSCs. Given the lack of increase in NOTCH family expression following enhanced NF-kB activation in either the CSC or non-CSC population (Supplementary Fig. S5), these results led us to hypothesize that the non-cell-autonomous function of NF-kB is mediated by

NOTCH ligands expressed in non-CSCs. The high-NF-kB cells showed enhanced JAG1 mRNA and protein expression; however, the expression levels were signicantly reduced in the low-NF-kB cells (Fig. 3b,c). TNFa stimulation also induced JAG1 mRNA and protein expression (Fig. 3d,e). Interestingly, the expression levels of other NOTCH ligands were not signicantly affected by NF-kB activation (Fig. 3b,d). Furthermore, TNFa stimulation did not induce the mRNA or protein expression of JAG1 in either the luminal-like or claudin-low cells, although the induction of RELB, a known NF-kB-inducible gene, was clearly observed (Fig. 3f,g).

These results indicate that JAG1 is the only NF-kB-regulated NOTCH ligand gene in the basal-like cell lines. Further, this NF-kB-dependent JAG1 expression is likely specic to the basal-like subtype.

JAG1 mediates NF-kB-mediated stimulation of CSCs by non-CSCs. To address whether JAG1 mediates trans signalling,

we rst generated basal-like cells overexpressing JAG1 (Fig. 4a). JAG1 overexpression resulted in an expansion of the CSC population (Fig. 4b). The co-culture of GFP cells with JAG1-overexpressing cells resulted in both CSC population expansion (Fig. 4c) and enhanced mammosphere formation (Fig. 4d) in the GFP cells. Furthermore, enhanced JAG1 expression elevated HEY1 and HEY2 expression (Fig. 4e). To further analyse the critical role of JAG1, we reduced endogenous JAG1 expression. CSC population expansion was signicantly reduced in the high-NF-kB cells in response to JAG1 silencing, whereas JAG1 silencing did not affect NF-kB activation, as evidenced by the efcient RELB induction (Fig. 4f,g). The TNFa-induced CSC population expansion was also signicantly reduced by JAG1 silencing (Fig. 4h,i). Moreover, the complementation of the JAG1-silenced cells with JAG1 abolished the shJAG1-mediated inhibition of TNFa stimulation-induced CSC expansion (Fig. 4j,k). These results clearly indicate that NF-kB-induced JAG1 expression in non-CSCs has a critical role in CSC expansion. To further conrm the requirement for NOTCH signalling in NF-kB-mediated trans signalling, we analysed the effect of g-secretase inhibitors (GSIs) and the knockdown of RBPJ, a component of the transcription complex that is essential for NOTCH target gene expression2830, on NF-kB-induced CSC expansion. Both the GSIs and RBPJ-silencing signicantly blocked the TNFa- and NF-kB-induced CSC population expansion (Fig. 4lq, Supplementary Fig. S6).

NF-kB-JAG1 axis is unique to the basal-like subtype in vivo. To determine whether the aforementioned functional link between NF-kB and NOTCH signalling in breast cancer cell lines can be applied to human breast cancer in vivo, 534 breast tumours from The Cancer Genome Atlas31 were categorized into basal-like, claudin-low, ERBB2-enriched and luminal-like subtypes based on a hierarchical clustering analysis of their gene expression proles (Supplementary Fig. S7). We then tested whether the NF-kB-target genes were highly expressed in the basal-like and claudin-low subtypes. Twenty-eight genes were chosen as NF-kB-dependent genes because their expression levels were induced by TNFa stimulation and repressed by IkBaSR expression in human HeLa cell lines32. A Gene Set Enrichment Analysis (GSEA)33,34 revealed that the NF-kB-dependent genes were highly expressed in the basal-like subtype when compared with the ERBB2-enriched and luminal-like subtypes, but this difference was not signicant when compared with the claudinlow subtype (Fig. 5a). Similarly, the NF-kB-dependent genes were highly expressed in the claudin-low subtype when compared with the ERBB2-enriched and luminal-like subtypes (Fig. 5b). These data strongly suggest that NF-kB is highly activated in basal-like and claudin-low subtype tumours, which is consistent with our ndings in the cell lines (Fig. 1a,b). We next investigated the relationship between NF-kB activation and JAG1 expression.

GSEA was performed with the NF-kB-dependent gene set and the set of genes whose expression levels were correlated with JAG1 expression in each subtype. The expression of the NF-kB-dependent gene set was signicantly correlated with

JAG1 expression (Fig. 5c), but not with the expression of other NOTCH ligands (Fig. 5d), in the basal-like subtype. These results strongly suggest that JAG1 is a unique gene that is specically regulated by NF-kB in basal-like breast tumours. Because CSCs are primarily responsible for the formation of tumour metastases, we next analysed the relationship between the expression levels of various NOTCH ligands and the metastasis-free survival rates using the same database (Supplementary Fig. S7). JAG1 expression was signicantly correlated with a short metastasis-free survival only in the basal-like breast tumour subtype (Fig. 5e). An

NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 5

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299

HCC1937

HCC1937 HCC1954

MB468

HCC1937

0 24

12 0 24

12 0 24 h

Cont

CSC fraction

Non-CSC fraction

HEY1 HEY2

IB IKK

HCC1937

HCC1954

***

HCC1954

***

Relative

gene expression

Relative

gene expression

***

*** ***

NS NS

2 *** *

3 * *

***

NS NS

0Virus: Cont IKK Cont IKK

JAG1

JAG2

DLL1

DLL3

DLL4

RELB

JAG1 JAG2 DLL1 DLL3 DLL4 RELB

10 ng ml1 TNF

***

0 h 2 h 12 h 24 h

***

Cont

IKK

Cont

IKK

Cont

IKK

***

NS NS

NS NS NS

JAG1 150

50 (kDa)

(kDa)

***

Relative

gene expression

Tubulin

JAG1

MB468

HCC1954

TNF:

JAG1

JAG2

DLL1

DLL3

DLL4

RELB

150

Tubulin

NS NS NS

*** ***

***

10 ng ml1 TNF (h)0 h 12 h 24 h

NS NS

Relative

gene expression

** *

NS NS

***

NS NS

Relative JAG1

expression

JAG1 JAG2 DLL1 DLL3 DLL4 RELB

Claudin-low

MB231 MB436

TNF:

JAG1

Tubulin

0 24

12 0 24 h

MB231 MCF7 T47D

MB436

Claudin-low

Luminal-like

JAG1

Tubulin

150

(kDa)

200 500

400

300

200

100

(kDa)

*** ***

***

Relative RELB

expression

6 *

Luminal-like

MCF7 T47D

100

TNF:

0 24

12 0 24 h

150

0 MB231 MB436

Claudin-low

0 MCF7 T47D

Luminal-like

Figure 3 | JAG1 is a unique NF-kB-inducible NOTCH ligand in basal-like breast cancers. (a) HEY1 and HEY2 mRNA expression levels in CSCs are higher than in the non-CSC population and are further enhanced by NF-kB activation in HCC1937 cells. Total RNA fractions were prepared from CSC and non-CSC cells, and a real-time reverse transcriptase PCR (RTPCR) analysis was performed (n 3, means.d.; Students t-test). (b) Real-time RTPCR

analysis of NOTCH ligands and RELB mRNA expression (n 3, means.d.; Students t-test) in control, IkBaSR and IKKb-expressing cells was performed in

two basal-like cell lines. (c) Western blotting analyses of JAG1 protein regulated by NF-kB. (d) Real-time PCR analysis of NOTCH ligands and of RELB mRNA expression (n 3, means.d.; Students t-test) in TNFa-treated basal-like subtype cells. (e) Western blotting analyses of JAG1 protein induced by

TNFa treatment. (f) Real-time PCR analysis of JAG1 and RELB mRNA expression (n 3, means.d.; Students t-test) in cells treated with 10 ng ml 1

TNFa for the indicated times. (g) Western blotting of JAG1 expression in cells treated with 10 ng ml 1 TNFa for the indicated times. All data are representative of three independent experiments. ND, not detected; NS, not signicant. *Po0.05, **Po0.01 and ***Po0.001 (a,b,d,f).

Extraction of Expression Module analysis35 of the same clinical data also revealed that the strong NF-kB activation and the NF-kB-JAG1 axis were specic to the basal-like subtype (Supplementary Fig. S8). The analyses of these clinical data strongly support our hypothesis that JAG1 has a basal-like sub-type-specic role in the maintenance of the CSC population.

NIK induces JAG1 expression in some breast cancer cell lines. Because TNFa stimulation induced JAG1 expression, JAG1 is

induced by the canonical NF-kB pathway. However, the expression of IkBaSR or IKKb altered the level of nuclear RELB, an NF-kB subunit activated by the non-canonical pathway, in addition to nuclear RELA (Fig. 6a). Moreover, we previously reported that basal-like cell lines show higher NIK mRNA expression levels than luminal-like cell lines and that enhanced NIK mRNA expression results in the accumulation and activation of NIK protein, leading to the constitutive activation of NF-kB5,36. Given that NIK is an activator of IKKa in the non-canonical pathway, these results led us to hypothesize that enhanced NIK expression-

6 NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299 ARTICLE

Cont JAG1

CD24/CD44high/

EpCAM+ CSC population

in GFP+ cells (%)

HCC1937

MB468

HCC 1937

HCC1954 HCC1937 MB468 HCC1954

HCC1954

MB468

HCC1954 15 1.5

20 1.6

1.2

0.8

0.4

JAG1

Cont

JAG1

Cont

CD24/CD44high/

EpCAM+ CSC

population (%)

JAG1

0 0 3 6 9 12

Days

*** * ***

Virus:

0.5

0 0

0.4

0.6

0.4

0.2

0.6

Cont

** ** ** **

** **

0.2

JAG1

Tubulin

150

50 (kDa)

Virus:

Cont

0 JAG1

Cont

JAG1

Cont

JAG1

0 3 6 9 12

Days

0 0 3 6 9 12

Days

HCC1937

HCC1954

HCC1937

12 * * ***

***

HCC1937 HEY1 HEY2

HCC1937

4 0.8

Sphere formation

(%)

0 Virus:

Virus:

Cont

Relative

gene expression

3 15

CD24/CD44high/

EpCAM+ CSC

population (%)

Cont IKK Virus: shLuc shJAG1 shLuc shJAG1

TNF: + + + +

RELB

Tubulin

JAG1

JAG1 150

50 (kDa)

Tubulin

150

** 0.6

0.4

0.2

50 (kDa)

Cont

JAG1

Virus: Virus:

0 Cont

JAG1

0 Cont

JAG1

shLuG

shJAG1

0 shLuG

shJAG1

Untreated TNF

Virus:shLuc shJAG1 30

Virus:

JAG1

Cont

TNF

CD24/CD44high/

EpCAM+ CSC

population (%)

Cont

JAG1

HCC1954

IKK

Cont

IKK

Cont

IKK

shLuc

shJAG1

shLuc

shJAG1

shLuc

shJAG1

shLuc

shJAG1

Virus:

Virus: Cont JAG1

JAG1

IKK

RELB

150

100 75

50 (kDa)

HCC1937

30 1.5

0.5

IKK

Cont

CD24/CD44high/

EpCAM+ CSC

population (%)

0 Virus:

shRBPJ

Tubulin

HCC1937

shLuc shJAG1 shLuc shJAG1

HCC1954 Untreated

shLuc

shJAG1

0 shLuc

shJAG1

Virus:

shLuc

No. 1 No. 2

shRBPJ

IKK

Cont

Virus: TNF:

RBPJ

Tubulin

shLuc

+ + +

IKK

Cont

IKK

No. 1 No. 2

50 (kDa)

JAG1 Untreated

** *

Cont

IKK

TNF

RBPJ

Tubulin

50 (kDa)

CD24/CD44high/EpCAM+

CSC population (%)

CD24/CD44high/EpCAM+

CSC population (%)

shLuc shRBPJ no. 1 shRBPJ no. 2

20 * **

***

CD24/CD44high/

EpCAM+ CSC

population (%)

DMSO

CD24/CD44high/

EpCAM+ CSC

population (%)

* *

DAPT

L685,458

DMSO

DAPT

L685,458

DMSO

DAPT

L685,458

DMSO

DAPT

DAPT L685,458

L685,458

DMSO

0Virus: Cont Cont

IKK

TNF

Figure 4 | JAG1 is crucial in the NF-kB-mediated stimulation of CSCs by non-CSCs. (a) Western blotting analyses of JAG1 expression in JAG1-overexpressing cells. (b) Flow-cytometry analysis of the CSC populations (n 3, means.d.; Students t-test) in JAG1-overexpressing cells.

(c,d) GFP-labelled cells were co-cultured with a 10-fold excess of control or JAG1-overexpressing cells. After the co-culture, the CSC populations (n 3,

means.d.; Students t-test) of the GFP-labelled cells were measured by ow-cytometry analysis (c). After 12 days of co-culture, the GFP-labelled cells were sorted and analysed for sphere-forming ability (n 3, means.d.; Students t-test) (d). (e) HEY1 and HEY2 mRNA expression (n 3, means.d.;

Students t-test) in JAG1-overexpressing cells. (f) Western blotting of whole-cell lysates derived from control or IKKb-overexpressing basal-like cells that additionally express either shLuc or shJAG1 (shLuc; control short hairpin RNA against luciferase gene). (g) Flow-cytometry analysis of the basal-like cells depicted in f (n 3, means.d.; Students t-test). (h) Western blotting analysis of whole-cell lysates derived from TNFa-treated basal-like cells that

express either shLuc or shJAG1. (i) Basal-like cells expressing either shLuc or shJAG1 were untreated or treated with TNFa10 ng ml 1 for 3 days, and then CSC populations (n 3, means.d.; Students t-test) were measured by ow-cytometry analysis. (j) Western blotting analyses of JAG1 expression in

shLuc- or shJAG1-expressing HCC1937 cells that were further complemented with JAG1. (k) The HCC1937 cells analysed in j were untreated or treated with TNFa (10 ng ml 1) for 3 days, and then CSC populations (n 3, means.d.; Students t-test) were measured by ow-cytometry analysis.

(l,m) Control, JAG1-expressing, IKKb-expressing (l) and TNFa-treated (m) HCC1937 cells were untreated or treated with N-[N-(3,5-diuorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (5 mM) or L685,458 (10 mM) for 3 days, and then CSC populations (n 3, means.d.; Students t-test) were

measured by ow-cytometry analysis. (n,o) Western blotting of whole-cell lysates derived from IKKb-overexpressing (n) and TNFa-treated (o) HCC1937 cells that further express shLuc or shRBPJ. (p,q) Flow-cytometry analysis of the HCC1937 cells depicted in n and o (n 3, means.d.; Students t-test).

All data are representative of three independent experiments. NS, not signicant. *Po0.05, **Po0.01 and ***Po0.001 (be,g,i,km,p,q).

induced non-canonical pathway activation may affect JAG1 expression. Because NIK protein is barely detectable due to its proteasome-mediated degradation37, we used proteasome inhibitor MG132 to stabilize NIK so that we could analyse the effect of NIK silencing. NIK silencing resulted in the signicant reduction of JAG1 expression in two basal-like cell lines, HCC1937 and HCC1143 (Fig. 6b,c). Furthermore, NIK

silencing resulted in the signicant reduction of the CSC population (Fig. 6d). Because NIK silencing resulted in the signicant reduction of nuclear RELB, but not RELA, in these two basal-like cell lines (Fig. 6e), JAG1 expression is likely regulated by the non-canonical pathway in addition to the canonical pathway. Therefore, the level of total NF-kB activation (the sum of canonical and non-canonical activation) may determine the level of JAG1

NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 7

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299

0.6 P = 0.030 P = 0.038 P = 0.81 P < 0.001

8,579

Claudin

Enrichment

score (ES)

0.40.2

0.60.40.2 0

0.20.1 0

0.1

0.60.40.2 0

1 0

0 1 1 2

Enrichment

score (ES)

P = 0.0084

0.60.40.2 0

Ranked list

metric

9,518 9,016 8,994

Ranked list

metric

8,820

Basal

Luminal Basal ERBB2 Basal

Claudin

Luminal

Claudin

Rank in ordered data set

Basal-like

Claudin-low

ERBB2-enriched

Luminal-like

0.4

Enrichment

score (ES)

0.60.40.2 0

P = 0.016

P = 0.31

P = 0.11

P = 0.30

0.4

0.2

0.4

0.2

Ranked list

metric

1.0

0.5 0

1.0

0.5

8,808

0.5

9,008 9,097 8,666

0.5

1.0

0.5

High Low

JAG1

High Low High Low High Low

ERBB2

JAG1 JAG1 JAG1

Rank in ordered data set

Basal-like

0.1

P = 0.65

0.3

0.2

0.5

Enrichment

score (ES)

0.2

P = 0.61 P = 0.021 P = 0.86

0.2

0.5

0.1 0.1

0.1

0.4

0.2 0.3

0.6

1.0

Ranked list

metric

1.00.5

0.5

8,418 8,948 8,444 9,496

High High

Low Low

JAG2

DLL1 High Low

DLL3 High Low

DLL4

Rank in ordered data set

Basal-like

Claudin-low

Luminal-like

ERBB2-enriched

P = 0.045 P = 0.24 P > 0.5 P = 0.443

Cumulative

metastasis-free survival

1.0

0.5

1.0

0.5

1.0

0.5

1.0

0.5

JAG1 low (n=45) JAG1 high (n=46)

JAG1 low (n=16) JAG1 high (n=16)

JAG1 low (n=41) JAG1 high (n=41)

JAG1 low (n=36) JAG1 high (n=36)

0 0 4 8 0 4 8 0 4 8 0 4 8

Years

Figure 5 | Correlation between NF-kB-JAG1 signalling and patient survival. (a) Strong NF-kB activation in basal-like subtype of breast cancer. A GSEA was performed to determine whether the NF-kB-dependent gene set reported previously32 (Supplementary Table S2) was highly expressed in the basal-like subtype of breast cancer compared with other subtypes. All of the genes (n 17,814) on the microarray were ranked according to their differential

expression levels across the two subtypes using a signal-to-noise metric. The P-values were determined by a random permutation test. (b) Strong NF-kB activation in the claudin-low subtype of breast cancer. GESA was performed as in a, except that expression of the NF-kB-dependent gene set in the claudin-low subtype of breast cancer was compared with that in the luminal-like and ERBB2-enriched subtypes of breast cancer. (c) JAG1 expression is signicantly correlated with NF-kB activation in a basal-like subtype-specic manner. GSEA was performed to determine whether the NF-kB-dependent gene set was correlated with JAG1 expression in various breast cancer subtypes. All of the genes on the microarray were ranked according to their correlation with JAG1 expression using the Pearsons correlation metric. The P-values were determined by a random permutation test. (d) Lack of positive correlation between NF-kB activation and the expression of other NOTCH ligands in the basal-like subtype of breast cancer. GSEA was used to determine whether the NF-kB-dependent gene set was correlated with the expression of each NOTCH ligand in the basal-like subtype of breast cancer. All of the genes on the microarray were ranked according to their correlation with the expression of each NOTCH ligand using the Pearsons correlation metric.

The P-values were determined by a random permutation test. (e) KaplanMeier curves showing the relation between cumulative metastasis-free survival and the expression levels of JAG1 in patients with various subtypes of breast cancer. Statistical differences between pairs of metastasis-free survival curves were calculated using a log-rank test. Numbers shown in the ranked list metric column of ad indicate the ranking of the gene whose ranking metric score is zero.

expression in basal-like cells. Consistent with this hypothesis, NIK silencing resulted in the signicant reduction of total NF-kB activation in the HCC1937 and HCC1143 cell lines (Fig. 6c,e), whereas NIK silencing minimally affected the levels of nuclear RELA and RELB, total NF-kB activation, JAG1 expression and the CSC population in the HCC38 cell line (Fig. 6eh), even though all three of these cell lines highly express the NIK gene5. Thus, the contribution of NIK to total NF-kB activation and

JAG1 induction varies among basal-like cell lines and, perhaps, among the clinical samples of the basal-like breast cancers.

We have previously reported that the treatment of luminal-like cells with a histone deacetylase inhibitor increased NIK mRNA expression, whereas the same treatment of basal-like cells did not show this effect36. Furthermore, histone H3 acetylation in the 50 regulatory region of the NIK promoter was signicantly higher in the two basal-like cell lines compared with luminal-like MCF7

8 NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299 ARTICLE

Nuclear extract

HCC1937

HCC1143

HCC1937

HCC1143

HCC1143 HCC38

Virus: Virus:

Cont

IKK

Cont

IKK

shNIK no. 1

shNIK shNIK

No. 1 No. 2

shLuc

shNIK no. 1

shNIK no. 2

shLuc

shNIK no. 2

shLuc + + +

No. 1 No. 2

MG132:

shLuc

+ + +

Virus:

RELA 50 75

100 (kDa)

RELB

PARP

NIK

JAG1

100

50 (kDa)

150

Tubulin

(kDa)

HCC1143 ****

** **

HCC1937

Virus:

shLuc

shNIK no. 1

shNIK no. 2

+ + +

CD24/CD44high/

EpCAM+ CSC

population (%)

shNIK no. 1

shNIK no. 2

shLuc

shNIK no. 1

shNIK no. 2

shLuc

shNIK no. 1

shNIK no. 2

MG132:

Virus:

shLuc

Virus:

(kDa)

NIK

100

Tubulin

RELB

PARP

Tubulin

Oct1

50 75

100

Nuclear extract

50 Whole-cell lysate

Nuclear extract

(kDa)

shLuc

shNIK no. 1

0 shNIK no. 2

0 shLuc

shNIK no. 1

shNIK no. 2

EMSA

RELA

shLuc

shNIK no. 1

shNIK no. 2

NSNS MCF7

***

CD24/CD44high/

EpCAM+ CSC

population (%)

ChIP/input (%)

1 0

HCC1937 HCC1143

Antibody: Cont AcH3

NF-B

JAG1

Tubulin

150

(kDa)

shLuc

0 shNIK no. 1

shNIK no. 2

Virus:

Figure 6 | NIK mediates JAG1 expression in a subset of basal-like breast cancer cells. (a) Western blotting analyses of RELA and RELB levels in nuclear extracts of control, IkBaSR- and IKKb-expressing HCC1937 and HCC1143 cells. (b) NIK expression in HCC1937 and HCC1143 cells expressing shLuc, shNIK no. 1 or shNIK no. 2. HCC1937 and HCC1143 cells expressing shLuc, shNIK no. 1 or shNIK no. 2 were either untreated or treated with MG132 (10 mM)

for 2 h. Whole-cell lysates prepared with or without MG132 treatment were subjected to western blotting. (c) Western blotting analyses of JAG1 expression in HCC1937 and HCC1143 cells expressing shLuc, shNIK no. 1 or shNIK no. 2. (d) The CSC populations (n 3, means.d.; Students t-test) were

determined by ow-cytometry analysis. (e) RELA and RELB expression and NF-kB activation in nuclear extracts of shLuc-, shNIK no. 1- or shNIKno. 2-expressing cells. Nuclear extracts were subjected to western blotting and EMSA. Whole-cell lysates were analysed as a positive control for detection of tubulin. (f) NIK expression in HCC38 cells expressing shLuc, shNIK no. 1 or shNIK no. 2. Cells expressing shLuc, shNIK no. 1 or shNIK no. 2 were either untreated or treated with MG132 (10 mM) for 2 h. Whole-cell lysates prepared with or without MG132 treatment were subjected to western blotting.

(g) Western blotting analyses of JAG1 expression in HCC38 cells expressing shLuc, shNIK no. 1 or shNIK no. 2. (h) The CSC populations (n 3, means.d.;

Students t-test) were determined by ow-cytometry analysis. (i) The histone H3 acetylation prole in the NIK promoter region (n 3, means.d.;

Students t-test) was analysed using a chromatin immunoprecipitation (ChIP) assay in luminal-like (MCF7) and basal-like (HCC1937 and HCC1143) cell lines. All data are representative of three independent experiments. NS, not signicant. **Po0.01 and ***Po0.001 (d,h,i).

cells (Fig. 6i). These results strongly suggest that in some of the basal-like cell lines, epigenetically induced NIK expression has a critical role in NF-kB-induced JAG1 expression in non-CSCs, which could lead to CSC population expansion. Therefore, the NIK-NF-kB-JAG1-NOTCH axis may establish an intratumoural niche that regulates the CSC population in a subset of basal-like breast tumours in vivo.

NOTCH expands CSC population only in the basal-like subtype. We next addressed whether normal cells that constitute tumour microenvironments express JAG1 in an NF-kB-dependent manner. First, the expression of JAG1 (but not other NOTCH ligands) was induced in macrophages either by lipopolysaccharide treatment or by the ectopic expression of IKKbEE (Fig. 7ad). Second, the expression of JAG1 (but not other NOTCH ligands) was induced in mammary broblasts either by TNFa treatment or by the ectopic expression of IKKbEE (Fig. 7eh). In normal mammary gland development, PR induces RANK ligand expression in luminal epithelial cells38,39. RANK ligand then stimulates RANK in mammary stem cells (MaSCs) and basal myoepithelial cells to induce NF-kB activation, which promotes rapid cell growth that ultimately leads to mammary gland development. Interestingly, the treatment of mice with medroxyprogesterone acetate (MPA, a synthetic PR derivative)

signicantly induced JAG1 expression in basal myoepithelial cells (Fig. 7i). This result strongly suggests that RANK-induced NF-kB activation results in the induction of JAG1 expression in basal myoepithelial cells.

To understand the effect of JAG1 expression on normal cells that constitute tumour microenvironments on CSC population regulation in breast cancer, we generated claudin-low and luminal-like cell lines that overexpressed JAG1. As observed in the JAG1-overexpressing basal-like cells, JAG1 overexpression resulted in the activation of NOTCH signalling as judged by the induction of NOTCH1 ICD (Figs 4e and 8a). However, NOTCH activation scarcely resulted in the expansion of the CSC population of the claudin-low and luminal-like cell lines when compared with that of the basal-like cell lines (Figs 4b and 8b). These results strongly suggest that the NOTCH-dependent CSC population expansion is specic to the basal-like subtype and that the tumour microenvironment generated by JAG1-expressing normal cells is likely to maintain the CSC population of basal-like breast cancer.

DiscussionThe involvement of NF-kB in the maintenance of the CSC population in breast cancer has been reported elsewhere1215. Inhibition of NF-kB or IKKa activation results in the reduction of

NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 9

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299

a b c i

***

10 ng ml1 LPS

***

Krt14

Krt18

Tnfsf11

Jag1

0 h 12 h 24 h

***

LPS (10 ng ml1)

0 12 24 h

150

50 (kDa)

10 ***

Virus:

Cont

IKK

160

***

JAG1

IKK

150

Relative gene

expression

*NS NS NS NS

***

Tubulin

100

50 (kDa)

Tubulin

1.5

0.5

1.5

0.5

Jag1

Jag2

Dll1

Dll3

Dll4

0 Nfkbia (l[afii9837]B[afii9825])

***

d e f

Virus: Cont IKK

IKK

Relative gene

expression

*** ***

***

2 12 24 h

JAG1

150

100

Relative gene expression

TNF (10 ng ml1)

Virus:

Cont

JAG1

IKK

***

Tubulin

150

50 (kDa)

Tubulin

50 (kDa)

Jag1

Jag2

Dll1

Dll3

Dll4

Nfkbia (l[afii9837]B[afii9825])

g h

10 ng ml1 TNF

4 **

** ** **

0 h 2 h 12 h 24 h

*** Virus:

NS NS

Relative gene

expression

Cont IKK

Relative gene

expression

Basal

Luminal

Basal

Luminal

Jag1

Dll1

Dll4

Nfkbia (l[afii9837]B[afii9825])

0 Jag1 Jag2 Dll1 Dll3 Dll4

0 Nfkbia (l[afii9837]B[afii9825])

Jag2

Dll3

Sham MPA

Figure 7 | NF-kB-dependent induction of JAG1 in cells of the tumour microenvironment. (ad) Western blotting of JAG1 in bone marrow-derived macrophages stimulated with 10 ng ml 1 lipopolysaccharide (LPS) (a) or with IKKbEE expression (b). Real-time reverse transcriptase PCR (RTPCR)

analysis of Notch ligands and IkBa (Nfkbia) mRNA expression (n 3, means.d.; Students t-test) in bone marrow-derived macrophages with LPS (c) or

with IKKbEE expression (d). (eh) Western blotting of JAG1 in immortalized mouse mammary broblast cells stimulated with 10 ng ml 1 TNFa (e) or with IKKbEE expression (f). Real-time RTPCR analysis of Notch ligands and IkBa (Nfkbia) mRNA expression (n 3, means.d.; Students t-test) in

immortalized mouse mammary broblast cells with 10 ng ml 1 TNFa (g) or with IKKbEE expression (h). (i) Mammary basal and luminal epithelial cells were isolated from 10-week-old female mice with or without in vivo MPA treatment for 3 weeks. A real-time RTPCR analysis was performed to quantify

Jag1, Krt14, Krt8 and Tnfsf11 mRNA expression (n 3, means.d.; Students t-test). The induction of Nfkbia shown in c, d, g and h indicates NF-kB activation.

All data are representative of three independent experiments. ND, not detected; NS, not signicant. *Po0.05, **Po0.01 and ***Po0.001 (c,d,gi).

Basal-like

HCC1937

Claudin-low

MB231 BT549

Luminal-like

MCF7 T47D

Dysregulation of NF-B signalling

Epigenetic alterations Other mechanisms

NIK overexpression

NF-B activation in non- CSCs

Differentiation

Cytokines (TNF-) Intratumoural microenvironments

JAG1

Cont

JAG1

Cont

JAG1

Cont

JAG1

Virus:

Cont

JAG1

Cont

JAG1

ICD

NOTCH1

RBPJ

Tubulin

150

100

50 50 (kDa)

JAG1

NOTCH

Self-renewal

JAG1

Basal-like Claudin-low

HCC1937 MB231 BT549

Luminal-like

MCF7 T47D

TNF

CSC population (%)

NS 1.5

0.5

ALDEFLUOR+

* 15

CSCs

JAG1 JAG1

Mammaryepithelial cells Macrophages

Mammaryfibroblasts Inflammation infection

Normal cell microenvironment

Progesterone

Pregnancy

Virus:

Cont

JAG1

Cont

JAG1

Cont

JAG1

Cont

JAG1

Cont

JAG1

Figure 8 | JAG1-induced NOTCH activation expands the CSC population in a basal-like subtype-specic manner. (a) Western blotting of whole-cell lysates derived from control or JAG1-overexpressing basal-like, claudin-low and luminal-like cells was performed. (b) The percentagesof the ALDEFLUOR CSC population (n 3, means.d.; Students t-test) in control or JAG1-overexpressing cells were analysed. NS, not signicant.

*Po0.05. (c) A model illustrating the NF-kB-mediated maintenance of the CSC population of basal-like breast cancer in vivo. See text for details. All data are representative of three independent experiments.

10 NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299 ARTICLE

the CSC population of luminal-like breast cancer-generated MMTV-ErbB2 transgenic mice. However, the precise mechanisms by which NF-kB regulates CSC population remain unclear. On the other hand, the cell-autonomous role of NOTCH signalling in the maintenance of breast CSCs has been demonstrated2527. Moreover, it has been reported that JAG1 is highly expressed in basal-like subtype40 and that JAG1 expression correlates with poor outcome in breast cancer41,42. Therefore, it is critical to elucidate the functional interactions that occur between NF-kB and

NOTCH signalling to regulate the CSC population. This study addresses this issue and demonstrates the non-cell-autonomous role of NF-kB in the activation of NOTCH signalling in CSCs.

We propose a model by which NF-kB regulates the CSC population in basal-like breast cancers (Fig. 8c). The constitutive activation of NF-kB in basal-like breast cancer is signicantly higher than that in the luminal-like subtype. This result could be attributed to enhanced NIK expression via subtype-specic epigenetic alterations5,36, some other dysregulation of NF-kB signalling caused by unknown mechanisms or inammatory cytokines secreted from inammatory inltrates, such as macrophages. The cell-autonomous elevation of NF-kB activation induces JAG1 expression in a basal-like subtypespecic manner. Although JAG1 induction occurs in both CSC and non-CSC populations, the vast majority occurs in non-CSC populations. Therefore, intratumoural niches, which are primarily composed of JAG1-presenting non-CSCs, are formed and stimulate NOTCH signalling in CSCs to promote CSC self-renewal (the intratumoural microenvironments in Fig. 8c). In addition, normal cells that constitute tumour microenvironments also express JAG1 in an NF-kB-dependent manner. The stimulation of Toll-like receptors, which is triggered by infection or inammation, induces JAG1 and inammatory cytokines, such as TNFa in macrophages. TNFa in turn stimulates JAG1 expression in mammary broblasts and cancer cells. Moreover, given that the MPA treatment of mice resulted in JAG1 induction in basal myoepithelial cells, the continuous production of PR during pregnancy may induce JAG1 expression in basal myoepithelial cells in humans, thus partly accounting for the increased incidence of pregnancy-associated breast cancer43. Therefore, in addition to non-CSC populations, JAG-expressing macrophages, broblasts and basal myoepithelial cells may form niches that regulate the self-renewal of CSCs in an NF-kB-dependent manner (the normal cell microenvironments in Fig. 8c). Moreover, the NOTCH-dependent expansion of the CSC population may be specic to the basal-like subtype. Although the mechanism by which this specicity occurs remains unknown, some of the NOTCH target genes critical for CSC expansion could be induced only in the basal-like subtype. Alternatively, some genes whose products inhibit CSC expansion may be suppressed by JAG1- or RBPJ-mediated signalling, as it has been reported that JAG1 suppresses the expression of NOTCH target genes by the nuclear translocation of the JAG1 cytoplasmic fragment generated by g-secretase-dependent processing44, and that RBPJ is also involved in the suppression of NOTCH target gene expression45.

Analyses of clinical data revealed that the NF-kB-JAG1-NOTCH axis-dependent regulation of the CSC population observed in various breast cancer cell lines likely operates in vivo. The NF-kB target genes are highly expressed in basal-like breast tumours when compared with luminal-like and ERBB2-enriched subtypes. Furthermore, JAG1 expression is signicantly correlated with the expression of the NF-kB-target genes only in basal-like breast tumours. These results strongly suggest the presence of NF-kB-mediated JAG1 expression in basal-like breast cancer in vivo. More importantly, JAG1 expression levels correlated well with the metastasis-free survival

rates of basal-like breast cancer patients, but not with those of patients with other breast cancer subtypes. These results suggest that the intratumoural NF-kB-JAG1-NOTCH axis has a crucial role in regulating the CSC population of basal-like breast tumours.

Several groups have recently proposed that basal-like breast cancer originates from the luminal progenitor cell population16,17,19, which exhibits higher NF-kB activation levels when compared with MaSCs and basal myoepithelial cells46. Therefore, NF-kB activation in basal-like breast cancer cells and luminal progenitors may be upregulated by similar, not-yet-identied mechanisms in addition to the enhanced NIK expression.

Two mechanisms have been reported for NF-kB-dependent JAG1 induction. First, NF-kB directly activates the JAG1 promoter47. Second, JAG1 expression is induced by signals activated by IL-6, whose expression is directly induced by NF-kB48. Although cellcell contact-independent effects exist between high-NF-kB cells and CSCs for the regulation of the CSC population, a neutralizing antibody against IL-6 or IL-8 was unable to abrogate the TNFa-induced or IKKb-mediated expansion of the CSC population. These results strongly suggest that JAG1 is a direct target gene of NF-kB. Therefore, the NF-kB-dependent inducibility of JAG1 may be attributed to the open chromatin structure of the JAG1 locus that is specically formed in the basal-like subtype.

In addition to elucidating the functional link between NF-kB and NOTCH signalling in regulating the CSC population, our study contributes to the development of therapeutic strategies. RANK-mediated NF-kB activation is essential for MaSC proliferation39; therefore, inhibiting NF-kB activation exerts profound effects on normal breast development, which is not signicantly affected by the inhibition of NOTCH signalling26. In addition, given that GSI treatment signicantly inhibits the NF-kB-mediated expansion of the CSC population, specic JAG1 targeting may be a promising approach. Further studies on niches that maintain CSCs must be performed to develop effective therapeutic strategies against basal-like breast cancers.

Methods

Plasmids and reagents. Human cDNAs encoding IkBaSR (S32A/S36A), IKKb, IKKbEE (S177E/S181E), JAG1, GFP and RFP cDNAs were generated by PCR and inserted into the retroviral vector pMXs, which was obtained from T. Kitamura (University of Tokyo, Japan). The antibodies used were as follows: anti-IKKb (1:1,000, no. 2684), anti-RELB (1:1,000, no. 4922), anti-NIK (1:1,000, no. 4994), anti-Cleaved NOTCH1 (1:1,000, no. 4147) and anti-RBPJ (1:1,000, no. 5313) (Cell Signalling Technology, Danvers, MA, USA); anti-acetyl-histone H3 (cat. no. 06-599), anti-p52 (1:1,000; cat. no. 05-361) and anti-tubulin (1:2,000; cat. no. CP06) (Millipore, Darmstadt, Germany); anti-Notch1 (1:1,000, sc-32745) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-human CD44-FITC (1:50; no. 560977), anti-human CD44-biotin (1:50; no. 555477), anti-human CD24-PE (1:50; no. 555428) and anti-human CD49f-PE/Cy5 (1:50; no. 551129) (BD Pharmingen, San Diego, CA, USA); anti-human EpCAM-PerPC/Cy5.5 (1:50; no. 324212) (Biolegend, San Diego, CA, USA); a mouse epithelial cell enrichment cocktail (1:20; a mixture of biotinylated antibodies against CD45, TER119 and CD31; STEMCELL Technologies, Vancouver, Canada); anti-mouse CD24-FITC (1:50; eBioscience, San Diego, CA, USA); and anti-TNFa (2 mg ml 1; AF-210-NA), anti-IL-6 (2 mg ml 1;

AF-227-NA) and anti-IL-8 (2 mg ml 1; AF-208-NA) (R&D Systems, Minneapolis, MN, USA). The reagents used were as follows: N-[N-(3,5-diuorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, L685,458 and MG132(Peptide Institute, Osaka, Japan); epidermal growth factor and basic broblast growth factor (BD Pharmingen); TNFa, TGFb, IL-6 and IL-8 (Sigma-Aldrich,

St Louis, MO, USA); 7-amino-actinomycin D (7AAD) (Millipore); streptavidin-ECD (1:10; Beckman Coulter, Fullerton, CA, USA); and slow-release MPA pellets (Innovative Research of America, Sarasota, FL, USA).

Cell culture and transfection. Human breast cancer cell lines were obtained from the Cell Resource Center for Biomedical Research, Tohoku University (MCF7), and purchased from the American Type Culture Collection (BT-549, HCC1143, HCC1937, HCC1954, HCC38, HCC70, MDA-MB-231, MDA-MB-436, MDA-MB-453, MDA-MB-468 and T-47D). Plat-E and Plat-A retrovirus packaging cells were kindly provided by Dr Kitamura (Institute of Medical Science, University of Tokyo,

NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 11

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299

Tokyo, Japan). All cell lines were cultured according to the recommendations of the supplier. For the retrovirus-mediated gene transfer, the packaging Plat-A and Plat-E cells were transfected with the pMX plasmids by the calcium phosphate method, and virus stocks were prepared by collecting the culture medium. Human basal-like breast cancer cell lines were infected with the virus stock in the presence of 6 mg ml 1 polybrene (Sigma-Aldrich) followed by selection with puromycin; the resultant cell pools were used.

EMSA and quantication of NF-kB activation. Cells were collected at 24 h after medium change. Nuclear extracts were prepared, and EMSAs were then performed as described previously49. Quantication of relative NF-kB activities in breast cancer cell lines has been described previously5. Briey, intensities of retarded bands corresponding to the NF-kB and DNA complex were measured with a

BAS2000 Bioimage analyser (Fuji Film, Tokyo, Japan), and the NF-kB activation level of each cell line was normalized to that of TNFa-stimulated Jurkat cells.

Identication of CSCs by ow-cytometry analysis. Breast cancer cells were stained with anti-human CD44-FITC, anti-human CD24-PE and anti-human EpCAM-PerPC/Cy5.5 antibodies in the presence of 7AAD (1 mg ml 1) in PBS with 1% FBS for 30 min at 4 C; they were then analysed using an Epics XL ow cytometer (Beckman Coulter, Brea, CA, USA). The CSC and non-CSC populations were sorted on a FACSAria ow cytometer (BD Biosciences, San Jose, CA, USA) after staining as described above. Aldehyde dehydrogenase (ALDH) activity was measured using the ALDEFLUOR kit (STEMCELL Technologies) as previously described21. Cells were incubated in ALDEFLUOR assay buffer containing 1 mM

ALDH substrate. As a negative control, each cell sample was stained under identical conditions with 15 mM of diethylaminobenzaldehyde, a specic ALDH inhibitor. After 30 min of incubation at 37 C, the cells were analysed using an Epics XL ow cytometer.

In vitro co-culture assay. GFP-labelled cell lines with intact NF-kB activation (1 104 cells) were mixed with an unlabelled corresponding cell line that expresses

IKKb, IkBaSR or Jag1 (1 105 cells) in six-well plates. Cells were then detached

from the plates and stained with anti-human CD44-biotin/streptavidin-ECD (1:10), anti-human CD24-PE (1:50) and anti-human EpCAM-PerPC/Cy5.5 (1:50) antibodies in the presence of 7AAD (1 mg ml 1) in PBS with 1% FBS for 30 min at 4 C; they were then analysed using an Epics XL ow cytometer (Beckman Coulter). GFP cells were sorted on a FACSAria ow cytometer (BD Biosciences).

Sphere-formation assay. Breast cancer cells were dissociated into single-cell suspensions by trypsinization. The cells (100 cells per ml in each well) were then seeded into 24-well ultralow-attachment plates (Corning, Corning, NY, USA) in Mammary Epithelial Basal Medium (Lonza, Walkersville, MD, USA) supplemented with 10 ng ml 1 epidermal growth factor, 10 ng ml 1 basic broblast growth factor and B27 (Life Technologies). After 7 days, the spheres (450 mm) were counted to determine the sphere-forming efciency. Under these conditions, approximately 90% of the mammospheres were derived from a single cell (Supplementary Fig. S3).

Tumorigenicity studies. Animal research complied with protocols approved by the Committee for Animal Experimentation of the Waseda University. Breast cancer cells were dissociated into single-cell suspensions by trypsinization. The indicated numbers of cells in 100 ml of RPMI1640 (Wako, Tokyo, Japan) containing 50% Matrigel (BD Biosciences) were injected into the fat pads of 7-week-old female NOD/SCID mice (Clea Japan, Kanagawa, Japan). Ten weeks after injection, the mice were euthanised and necropsied, and the formed tumours were counted.

Western blotting. Whole-cell lysates were separated by SDSpolyacrylamide gel electrophoresis and transferred to polyvinylidene diuoride membranes (Immobilon P; Millipore). The membranes were then incubated with primary antibodies. Immunoreactive proteins were visualized with anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (GE Healthcare, Piscataway, NJ, USA), which was followed by processing with an ECL detection system (GE Healthcare). Full-length images of immunoblots are shown in Supplementary Fig. S9.

Quantitative real-time reverse transcriptase PCR assays. Total RNA was isolated with the Trizol reagent (Life Technologies). cDNA was synthesized from 1 mg of total RNA with Prime Script II (Takara Bio, Shiga, Japan). Quantitative real-time PCR analysis was performed on a Life Technologies 7300 Fast Real-Time PCR System using FastStart Universal SYBR Green Master Mix (Roche, Basel, Switzerland). The level of b-actin expression was used to normalize the data. The primers used for real-time reverse transcriptase PCR are described in Supplementary Table S1.

Chromatin immunoprecipitation assay. A chromatin immunoprecipitation assay was performed as described previously36. Anti-acetyl-histone H3 was used for immunoprecipitation. The immunoprecipitation efciency of the NIK promoter region ( 263 to 152) was analysed by real-time PCR using a FastStart Universal

SYBR Green Master (Roche) according to the manufacturers instructions. The primers used were 50-ACATCGTCCGGAAATAGTGC-30 and 50-CCTACGCCAACCAATGAGAC-30.

Preparation of primary mouse cells. The preparation of bone marrow-derived macrophages was performed as described previously50. Immortalized mouse mammary broblast cells were established from primary mouse mammary broblast cells via the retrovirus-mediated expression of SV40 Large T antigens. Cells were cultured in DMEM (Nissui, Tokyo, Japan) supplemented with 10% FBS. For the analysis of Jag1 mRNA expression in primary mammary epithelial cells, slow-release MPA pellets were implanted into 7-week-old female C57/B6J mice (Japan SLC, Hamamatsu, Japan) as previously described38. Three weeks after implantation, cells were isolated from the mouse mammary fat pads as previously described and stained with a mouse epithelial cell enrichment cocktail (a mixture of biotinylated antibodies against CD45, TER119 and CD31), streptavidin-ECD, anti-mouse CD24-FITC, anti-human CD49f-PE/Cy5 and 1 mg ml 1 7AAD. The cells were sorted, with the FACSAria LineageCD24highCD49fmid cells as the luminal epithelial cells and the LineageCD24midCD49fhigh cells as the basal epithelial cells.

Construction of the short hairpin RNA expression vector. The target sequence 50-GGAGCACATTTGCAGTGAATT-30 was used to silence human JAG1 expression; 50-GCTGGAAGACAGTACTTTAGA-30 (shRBPJ no. 1) and 50-GCACGTTAGGTGGTTAAATCA-30 (shRBPJ no. 2) were used for human RBPJ expression; 50-GGTGTGAAAGTCCAAATACAG-30 (shNIK no. 1) and 50-GGAGGAATACCTAGTGCATGC-30 (shNIK no. 2) were used for human NIK expression; and the rey luciferase-targeted sequence 50-GATTTCGAGTCGTCTTAATGT-30 was used as a control. A construct comprising the sense and anti-sense sequences with an intervening 11-bp loop sequence (50-GTGTGCTGTCC-30) was inserted between the BglII and HindIII sites of the pSUPER.retro.puro vector (OligoEngine, Seattle, WA, USA).

Clinical analysis. A hierarchical clustering analysis of publicly available cDNA microarray data derived from 534 primary mammary tumour samples (tcga-data.nci.nih.gov/; The Cancer Genome Atlas)31 was performed using the GenePattern software program (http://www.broad.mit.edu/cancer/software/genepattern

Web End =www.broad.mit.edu/cancer/software/genepattern; Broad Institute, MA, USA)51. Each cluster was classied by the marker gene expression of each breast cancer subtype. A GSEA (http://www.broadinstitute.org/gsea/index.jsp

Web End =www.broadinstitute.org/gsea/ http://www.broadinstitute.org/gsea/index.jsp

Web End =index.jsp ; Broad Institute)33,34 and Extraction of Expression Module analysis35 were performed for each breast cancer subtype with the NF-kB-dependent gene set TIAN_TNF_SIGNALING_VIA_NFKB (http://www.broadinstitute.org/gsea/msigdb/cards/TIAN_TNF_SIGNALING_VIA_NFKB.html

Web End =www.broadinstitute.org/gsea/msigdb/ http://www.broadinstitute.org/gsea/msigdb/cards/TIAN_TNF_SIGNALING_VIA_NFKB.html

Web End =cards/TIAN_TNF_SIGNALING_VIA_NFKB.html ; Broad Institute)32.

Statistics. Statistically signicant differences between the mean values were determined using a two-tailed Students t-test and Fishers exact test. All data are representative of three independent experiments. The values represent the means of triplicate sampless.d. P-values o0.05 were considered statistically signicant.

References

1. Vargo-Gogola, T. & Rosen, J. Modelling breast cancer: one size does not t all. Nat. Rev. Cancer 7, 659672 (2007).

2. Herschkowitz, J. I. et al. Identication of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome. Biol. 8, R76 (2007).

3. Sorlie, T. et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc. Natl Acad. Sci. USA 100, 84188423 (2003).

4. Rakha, E., Reis-Filho, J. & Ellis, I. Basal-like breast cancer: a critical review.J. Clin. Oncol. 26, 25682581 (2008).5. Yamaguchi, N. et al. Constitutive activation of nuclear factor-kB is preferentially involved in the proliferation of basal-like subtype breast cancer cell lines. Cancer Sci. 100, 16681674 (2009).

6. Hayden, M. S. & Ghosh, S. Shared principles in NF-kB signaling. Cell 132, 344362 (2008).

7. Hacker, H. & Karin, M. Regulation and function of IKK and IKK-related kinases. Sci. STKE 2006, re13 (2006).

8. Sun, S. C. Controlling the fate of NIK: a central stage in noncanonical NF-kB signaling. Sci. Signal 3, pe18 (2010).

9. Perkins, N. D. Integrating cell-signalling pathways with NF-kB and IKK function. Nat. Rev. Mol. Cell Biol. 8, 4962 (2007).

10. Dalerba, P., Cho, R. W. & Clarke, M. F. Cancer stem cells: models and concepts. Annu. Rev. Med. 58, 267284 (2007).

11. Visvader, J. E. & Lindeman, G. J. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat. Rev. Cancer 8, 755768 (2008).

12 NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3299 ARTICLE

12. Cao, Y., Luo, J. L. & Karin, M. IkB kinase a kinase activity is required for self-renewal of ErbB2/Her2-transformed mammary tumor-initiating cells. Proc. Natl Acad. Sci. USA 104, 1585215857 (2007).

13. Liu, M. et al. The canonical NF-kB pathway governs mammary tumorigenesis in transgenic mice and tumor stem cell expansion. Cancer Res. 70, 1046410473 (2010).

14. Zhou, J. et al. NF-kB pathway inhibitors preferentially inhibit breast cancer stem-like cells. Breast Cancer Res. Treat. 111, 419427 (2008).

15. Murohashi, M. et al. Gene set enrichment analysis provides insight into novel signalling pathways in breast cancer stem cells. Br. J. Cancer 102, 206212 (2010).

16. Lim, E. et al. Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat. Med. 15, 907913 (2009).

17. Molyneux, G. et al. BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells. Cell Stem Cell 7, 403417 (2010).

18. Visvader, J. E. Cells of origin in cancer. Nature 469, 314322 (2011).19. Keller, P. J. et al. Dening the cellular precursors to human breast cancer. Proc. Natl Acad. Sci. USA 109, 27722777 (2012).

20. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J. & Clarke, M. F. Prospective identication of tumorigenic breast cancer cells. Proc. Natl Acad. Sci. USA 100, 39833988 (2003).

21. Ginestier, C. et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555567 (2007).

22. Charafe-Jauffret, E. et al. Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res. 69, 13021313 (2009).

23. Balkwill, F. Tumour necrosis factor and cancer. Nat. Rev. Cancer 9, 361371 (2009).

24. Iliopoulos, D. et al. An epigenetic switch involving NF-kB, Lin28, Let-7 microRNA, and IL6 links inammation to cell transformation. Cell 139, 693706 (2009).

25. Harrison, H. et al. Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor. Cancer Res. 70, 709718 (2010).

26. Bouras, T. et al. Notch signaling regulates mammary stem cell function and luminal cell-fate commitment. Cell Stem Cell 3, 429441 (2008).

27. Dontu, G. et al. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res. 6, R605R615 (2004).28. Bray, S. J. Notch signalling: a simple pathway becomes complex. Nat. Rev. Mol. Cell Biol. 7, 678689 (2006).

29. Iso, T. et al. HERP, a new primary target of Notch regulated by ligand binding. Mol. Cell Biol. 21, 60716079 (2001).

30. Iso, T. et al. HES and HERP families: multiple effectors of the Notch signaling pathway. J. Cell Physiol. 194, 237255 (2003).

31. Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 490, 6170 (2012).

32. Tian, B., Nowak, D. E., Jamaluddin, M., Wang, S. & Brasier, A. R. Identication of direct genomic targets downstream of the nuclear factor-kB transcription factor mediating tumor necrosis factor signaling. J. Biol. Chem. 280, 1743517448 (2005).

33. Mootha, V. K. et al. PGC-1a-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat. Genet. 34, 267273 (2003).

34. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression proles. Proc. Natl Acad. Sci. USA 102, 1554515550 (2005).

35. Niida, A. et al. Gene set-based module discovery in the breast cancer transcriptome. BMC Bioinformatics 10, 71 (2009).

36. Yamamoto, M. et al. Epigenetic alteration of the NF-kB-inducing kinase (NIK) gene is involved in enhanced NIK expression in basal-like breast cancer. Cancer Sci. 101, 23912397 (2010).

37. Liao, G. et al. Regulation of the NF-kB-inducing kinase by tumor necrosis factor receptor-associated factor 3-induced degradation. J. Biol. Chem. 279, 2624326250 (2004).

38. Joshi, P. A. et al. Progesterone induces adult mammary stem cell expansion. Nature 465, 803807 (2010).

39. Asselin-Labat, M. L. et al. Control of mammary stem cell function by steroid hormone signalling. Nature 465, 798802 (2010).

40. Reedijk, M. et al. JAG1 expression is associated with a basal phenotype and recurrence in lymph node-negative breast cancer. Breast Cancer Res. Treat. 111, 439448 (2008).

41. Reedijk, M. et al. High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival. Cancer Res. 65, 85308537 (2005).

42. Dickson, B. C. et al. High-level JAG1 mRNA and protein predict poor outcome in breast cancer. Mod. Pathol. 20, 685693 (2007).

43. Lambe, M. et al. Transient increase in the risk of breast cancer after giving birth. N. Engl. J. Med. 331, 59 (1994).

44. LaVoie, M. J. et al. The Notch ligands, Jagged and Delta, are sequentially processed by a-secretase and presenilin/g-secretase and release signaling fragments. J. Biol. Chem. 278, 3442734437 (2003).

45. Kao, H. Y. et al. A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev. 12, 22692277 (1998).

46. Pratt, M. A. et al. The canonical NF-kB pathway is required for formation of luminal mammary neoplasias and is activated in the mammary progenitor population. Oncogene 28, 27102722 (2009).

47. Johnston, D. A., Dong, B. & Hughes, C. C. TNF induction of jagged-1 in endothelial cells is NFkB-dependent. Gene 435, 3644 (2009).

48. Sansone, P. et al. IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland. J. Clin. Invest. 117, 39884002 (2007).

49. Yamaguchi, N. et al. NOTCH3 signaling pathway plays crucial roles in the proliferation of ErbB2-negative human breast cancer cells. Cancer Res. 68, 18811888 (2008).

50. Gohda, J. et al. RANK-mediated amplication of TRAF6 signaling leads to NFATc1 induction during osteoclastogenesis. EMBO J. 24, 790799 (2005).

51. Reich, M. et al. GenePattern 2.0. Nat. Genet. 38, 500501 (2006).

Acknowledgements

We thank T. Akiyama, J. Gohda and A. Niida for their helpful input and K. Miyazaki for secretarial assistance. This work was partially supported by a grant-in-aid for Scientic Research on Innovative Areas and by a contract research fund for the Program of Japan Initiative for Global Research Network on Infectious Diseases from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (J-i.I.), by the Takeda Science Foundation (J.-i.I.). M.Y. is a research fellow of the Japan Society for the Promotion of Science.

Author contributions

All authors contributed to the experimental design and data interpretation. M.Y. and Y.T. performed the experiments. M.Y., T.I.-K., K.S., N.Y. and J.-i.I. planned the project. M.Y. and J.-i.I. wrote the paper.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Web End =http://www.nature.com/ http://www.nature.com/naturecommunications

Web End =naturecommunications

Competing nancial interests: The authors declare no competing nancial interests.

Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/

Web End =http://npg.nature.com/ http://npg.nature.com/reprintsandpermissions/

Web End =reprintsandpermissions/

How to cite this article: Yamamoto, M. et al. NF-kB non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype. Nat. Commun. 4:2299 doi: 10.1038/ncomms3299 (2013).

NATURE COMMUNICATIONS | 4:2299 | DOI: 10.1038/ncomms3299 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 13

Word count: 11710

Show less

Abstract

Translate

Patients with triple-negative breast cancer display the highest rates of early relapse of all patients with breast cancer. The basal-like subtype, a subgroup of triple-negative breast cancer, exhibits high levels of constitutively active NF-κB signalling. Here we show that NF-κB activation, induced by inflammatory cytokines or by epigenetically dysregulated NIK expression, cell-autonomously upregulates JAG1 expression in non-cancer stem cells. This upregulation stimulates NOTCH signalling in cancer stem cells in trans, leading to an expansion of cancer stem cell populations. Among breast cancers, the NF-κB-dependent induction of JAG1 and the NOTCH-dependent expansion of the cancer stem cell population occur only in the basal-like subtype. Collectively, our results indicate that NF-κB has a non-cell-autonomous role in regulating cancer stem cell populations by forming intratumoural microenvironments composed of JAG1-expressing non-cancer stem cells with a basal-like subtype.

Details

Title

NF-[kappa]B non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype

Author

Yamamoto, Mizuki; Taguchi, Yuu; Ito-kureha, Taku; Semba, Kentaro; Yamaguchi, Noritaka; Inoue, Jun-ichiro

Pages

2299

Publication year

2013

Publication date

Aug 2013

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms3299

ProQuest document ID

1419377822

NF-[kappa]B non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype

Jump to:

Full text

Abstract

Details

Suggested sources