Department of Orthopaedic Surgery, Johns Hopkins...

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CITED2 Modulates Breast Cancer Metastatic Ability Through Effects on IKKα.

Swaathi Jayaraman, Michele Doucet, Wen Min Lau and Scott L. Kominsky

Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore,

MD.

Running title: CITED2 modulates human breast cancer metastasis

Keywords: CITED2, IKKα, invasion, metastasis, cancer

Financial support: Research reported in this publication was supported by the National Cancer

Institute of the National Institutes of Health under Award Number R01CA157687. The content

is solely the responsibility of the authors and does not necessarily represent the official views of

the National Institutes of Health.

Address correspondence to: Scott L. Kominsky, Ph.D., 720 Rutland Avenue, Ross 232,

Baltimore, MD 21205. Fax: 410-502-6414; E-mail: [email protected]

The authors disclose no potential conflicts of interest.

on September 6, 2018. © 2016 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 23, 2016; DOI: 10.1158/1541-7786.MCR-16-0081

http://mcr.aacrjournals.org/

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Abstract

Previously, we identified the transcriptional co-activator CITED2 as a potential facilitator

of bone metastasis using a murine mammary cancer model. Extending these studies to human

breast cancer, it was observed that CITED2 mRNA expression was significantly elevated in

patient specimens of metastatic breast cancer relative to primary tumors, with highest levels in

metastasis to bone relative to non-bone sites. To further evaluate CITED2 functions in breast

cancer metastasis, CITED2 expression was stably reduced in the human breast cancer cell lines

MDA-MB-231 and MDA-MB-468, which are metastatic in animal models. While CITED2

knockdown had no effect on cell proliferation, cell migration and invasion were significantly

reduced, as was the establishment of metastasis following intra-cardiac administration in athymic

nude mice. To explore the mechanism behind these effects, gene expression following CITED2

knockdown in MDA-MB-231 cells by cDNA microarray was performed. As confirmed at the

mRNA and protein levels in both MDA-MB-231 and MDA-MB-468 cells, expression of the NF-

κB regulator IKKα was significantly reduced along with several NF-κB targets with known roles

in metastasis (OPN, MMP9, uPA, SPARC, IL-11 and IL-1β). Further, ChIP assay revealed

recruitment of CITED2 to the promoter of IKKα, indicating a direct role in regulating its

expression. Consistent with reduced IKKα expression, CITED2 knockdown inhibited both

canonical and non-canonical NF-κB signaling. Finally, restoration of IKKα expression following

CITED2 knockdown in MDA-MB-231 and MDA-MB-468 cells rescued their invasive ability.

Collectively, these data demonstrate that CITED2 modulates metastatic ability in human breast

cancer cells, at least in part, through the regulation of IKKα.




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Implications: The current study highlights the role of CITED2 in facilitating breast cancer

metastasis, partly via regulation of IKKα.

Introduction

Breast cancer is the most frequently diagnosed cancer in women worldwide and the

second most commonly occurring cancer overall. While primary tumors may be effectively

treated when detected early, metastatic disease is largely incurable and represents the ultimate

cause of mortality in breast cancer patients. It is estimated that ~6% of patients already have

metastatic disease at the time of diagnosis while ~20-50% of patients who are initially diagnosed

with early stage breast cancer will eventually develop metastasis (1). Sadly, the median survival

time for patients with metastatic breast cancer is only 18-30 months. Despite recent research

efforts, elucidation of the critical drivers of metastasis and their mechanism of action is lacking.

Filling this knowledge gap is essential to the development of novel therapeutic modalities and

improving the clinical management of this disease.

The Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2

(CITED2) is a non-DNA binding transcriptional co-activator that was originally discovered for

its role in development (2-5). As a transcriptional co-activator, CITED2 interacts with several

transcription factors such as p300/CBP, Lhx2, TFAP2, Smad2/Smad3, PPARγ and estrogen

receptor, modulating their ability to activate gene transcription (6-11). Beyond its involvement in

development, CITED2 has also been reported to play a role in cancer, including that of the skin,

colon and lung (12-14). Recently, we identified CITED2 as a potential facilitator of breast cancer

bone metastasis using a murine mammary cancer model (15). While our preliminary analysis of

primary human breast tumor tissues revealed significantly higher levels of CITED2 mRNA




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relative to normal mammary epithelium (15), its expression pattern in metastatic lesions and

functional contribution to human breast cancer metastasis remain unclear.

In this study, we investigated the role of CITED2 in human breast cancer metastasis.

Here, we show that in breast cancer patients, CITED2 expression is significantly elevated in

metastatic lesions relative to primary tumors, with highest levels in bone metastasis. Further,

utilizing two highly invasive breast cancer cell lines, we show that stable knockdown of CITED2

significantly reduces tumor migration and invasion in vitro and the establishment of metastasis in

vivo. Lastly, we provide evidence that CITED2 mediates metastatic ability in human breast

cancer cells, at least in part, by regulating the expression of IKKα.

Materials and Methods

Cell lines, tissues and treatment

The human breast cancer cell lines MDA-MB-231 and MDA-MB-468 were obtained

from American Type Culture Collection, Rockville, MD (2014) and were authenticated using

DNA profiling and cytogenetic analysis by the cell bank. Cells were utilized for the experiments

within 6 months from the time of resuscitation. MDA-MB-231 cells were maintained in RPMI

medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals) and

MDA-MB-468 cells were maintained in DMEM medium (Gibco) supplemented with 10% FBS

and 1% L-Glutamine (Gibco). To identify genes that are regulated by the NF-κB pathway, cells

were treated with 10 µM of PS1145 (Sigma-Aldrich) for 16 hours at 37°C.

Normal mammary epithelium samples, kindly provided by Dr. Saraswati Sukumar (Johns

Hopkins University School of Medicine, Baltimore, MD), were prepared from reduction

mammoplasty specimens of women with no breast abnormalities. Normal and tumor tissues were




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obtained from the Surgical Pathology Division of the Johns Hopkins Hospital following the

approval of the institutional review board (IRB) of the Johns Hopkins University School of

Medicine. For all specimens, required written informed patient consents were obtained as

approved by the IRB.

Transfection

To study the effects of CITED2 in human breast cancer metastasis, MDA-MB-231 and

MDA-MB-468 cells were infected with the lentiviral shRNA expression vector pLKO.1-puro

(Addgene plasmid 8453) containing siRNA sequence specific for scrambled or CITED2. The

CITED2 siRNA sequence has been described previously (9, 16). Stable cells were selected in the

presence of 1 µg/ml puromycin (Sigma-Aldrich) for one week and utilized for subsequent

experiments.

For experiments involving re-expression of IKKα, shCITED2-expressing cells were

transiently transfected with a 3:1 ratio of Xtreme gene HP DNA transfection reagent (Roche) and

pCR3.1-FLAG-IKKα vector [a kind gift from Hiroyasu Nakano (Addgene plasmid 15467) (17)]

or empty vector in OPTI-MEM medium (Gibco) for 24, 48 or 72 hours.

Quantitative (q)RT-PCR

Total RNA from tissue samples and cell lines was extracted using Trizol (Invitrogen) and

cDNA was generated using a reverse transcription system (Promega). The qRT-PCR parameters

have been described previously (11). Amplification of 36B4 was used as an internal control.

Relative expression between samples was calculated by the comparative CT method. The primer

sequences used were: CITED2 (sense) 5’-ACCATCACCCTGCCCACC-3’, (antisense)




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CGTAGTGTATGTGCTCGCCCA; IKKα (sense) 5’-GGCTTCGGGAACGTCTGTC-3’,

(antisense) 5’-TTTGGTACTTAGCTCTAGGCGA-3’; OPN (sense) 5’-

GAAGTTTCGCAGACCTGACAT-3’, (antisense) 5’-GTATGCACCATTCAACTCCTCG-3’;

MMP9 (sense) 5’-GGGACGCAGACATCGTCATC-3’, (antisense) 5’-

TCGTCATCGTCGAAATGGGC-3’; uPA (sense) 5’-GGGAATGGTCACTTTTACCGAG-3’,

(antisense) 5’-GGGCATGGTACGTTTGCTG-3’; SPARC (sense) 5’-

AGCACCCCATTGACGGGTA-3’, (antisense) 5’-GGTCACAGGTCTCGAAAAAGC-3’; IL-11

(sense) 5’-CGAGCGGACCTACTGTCCTA-3’, (antisense) 5’-

GCCCAGTCAAGTGTCAGGTG-3’; IL-1β (sense) 5’-ATGATGGCTTATTACAGTGGCAA-

3’, (antisense) 5’-GTCGGAGATTCGTAGCTGGA-3’; 36B4 (sense) 5’-

GAAGGCTGTGGTGCTGATGG-3’, (antisense) 5’-CCCCTGGAGATTTTAGTGGT-3’.

Immunohistochemistry

Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene

(Fisher Scientific) and rehydrated through a graded series of ethanol (Pharmco-AAPER).

Sections were immersed in antigen retrieval solution (Dako) and heated in a steamer for 20

minutes. Cooled sections were washed with phosphate buffer saline (PBS, Gibco) and

endogenous peroxidase activity was quenched by immersing sections in 3% hydrogen peroxide

(Fischer Scientific) for 12 minutes and washed with PBS. Sections were blocked by incubation

with protein block solution (Dako) for 30 minutes at room temperature and incubated at 4°C for

18 hours with goat anti-CITED2 (1:500; Everest Biotech). Sections were then sequentially

incubated for 15 minutes at room temperature with streptavidin-biotin complex, Tyramide

amplification reagent and streptavidin-horse radish peroxidase (HRP) from the DACO CSA Kit




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(Vector Laboratories). To visualize proteins, the chromogen 3, 3-diaminobenzamindine (DAB;

Open Biosystems) was added for two minutes at room temperature. Sections were subsequently

washed in water and counterstained with hematoxylin Gill No. 3 (Sigma-Aldrich).

Western analysis

Total protein extracts from cell lines were obtained as previously described (15).

Cytoplasmic and nuclear extracts was processed using NE-PER cytoplasmic and nuclear

extraction reagents (Thermo Scientific) according to manufacturer’s instructions. Conditioned

medium was collected by maintaining the cells in serum free medium. Samples were resolved

using SDS-PAGE, transferred to nitrocellulose membrane (Bio-Rad) and probed with sheep anti-

CITED2 (1:250; R&D Systems), rabbit anti-IKKα, anti-IκBα, anti-p65, anti-RelB, anti-HDAC1

(1:1000; Cell Signaling Technology), mouse anti-GAPDH (1:10,000; kindly provided by Dr.

Shanmugasundaram Ganapathy Kanniappan, Johns Hopkins University School of Medicine,

Baltimore, MD) or Actin (1:1000; Sigma-Aldrich) antibodies. Membranes were incubated with

horseradish peroxidase-conjugated antibody against sheep (1:2000; R&D Systems), rabbit or

mouse (1:2000; GE HealthCare) IgG and binding was revealed by chemiluminescence detection

(Millipore).

Proliferation assay

The in vitro proliferation of cells was determined by MTS assay using 0.2 mg/ml MTS

reagent (Promega) as previously described (15). Data for each time point was obtained in

triplicate per experimental condition.




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Migration and invasion assay

A 24 well plate containing either 8.0 μm pore cell culture insert (BD Falcon) or 8.0 μm

pore transwell-inserts pre-coated with 100 µl Matrigel (BD Falcon) was utilized for the

migration and invasion assays, respectively. Tumor cells (2.5 x 104 cells) were seeded in the

upper chamber in medium containing 0% FBS and the bottom chamber filled with medium

containing either 0% or 20% FBS as the chemo-attractant. After 16 hours (in case of migration)

or 48 hours (in case of invasion), cells in the upper chamber were removed with cotton swabs.

Cells that migrated or invaded to the lower surface of the insert were fixed in 100% cold

methanol (Fischer Scientific), washed in PBS and stained with 2% crystal violet (Harleco). Three

representative images from each well were captured at 100X magnification by light microscopy

and the total number of migrated or invaded cells per image was counted using ImageJ imaging

software (National Institute of Health, Bethesda, MD). Data are representative of at least two

independent experiments performed in triplicates per experimental condition.

In vivo assessment of breast cancer metastasis

Tumor cells (1 x 105 cells) from each group were injected into the left cardiac ventricle of

five week old athymic nude mice (Taconic) [For MDA-MB -231 cells, n = 9 (scramble) and 10

(shCITED2); For MDA-MB-468 cells, n = 10 (scramble) and n = 7 (shCITED2)]. Two weeks

later, the establishment of tumor-induced osteolysis in the bone was analyzed by obtaining

digital radiographic images of the femur and tibia twice a week using a Faxitron MX-20 X-ray

unit (Faxitron X-ray Corp.) until termination of the experiment. The experiment was terminated

when animals became moribund. The osteolytic area in the radiographic images was measured

using MetaMorph image analysis software (Meta Imaging Series version 6.1, Universal Imaging




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Corp.). Tumor lesions within the bone were analyzed by H&E staining of bone sections

decalcified in 10% EDTA (Sigma-Aldrich). Brain, liver and lungs were harvested and

maintained in Bouin’s fixative (RICCA chemical) for 24 hours and counted for the total number

of macro-metastatic lesions.

All animal experiments were carried out in accordance with the National Research

Council’s ‘‘Guide to the Care and Use of Laboratory Animals’’. Animal use was approved by

the Johns Hopkins Animal Care and Use Committee, animal welfare assurance #A3272-01,

protocol #MO10M450.

Microarray analysis

cDNA expression between scramble and shCITED2 MDA-MB-231 cells was compared

using Agilent Human GE 4x44K v2 microarray (G4845A). Log2 transformed signal intensities,

without background subtraction were imported into GeneSpring GX 10 software (Agilent

Technologies) and (quantile) normalized within the sample type. Differentially expressed genes

in shCITED2-expressing cells relative to scramble cells were identified based on ≥ two-fold

change in gene expression. Quality assessment of samples and microarray analysis were

conducted at the Sidney Kimmel Cancer Center Microarray Core Facility at Johns Hopkins

University School of Medicine, Baltimore, MD (supported by NIH grant P30 CA006973 entitled

Regional Oncology Research Center). Microarray data are deposited in the Array Express

database www.ebi.ac.uk/arrayexpress under accession number E-MTAB-4267.

ELISA analysis




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OPN and IL-11 ELISA immunoassay (R&D Systems) were performed on serum free

tumor-conditioned media obtained from cell lines according to manufacturer’s instructions.

Electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA) was performed on nuclear cell lysates using

the LightShift EMSA and Chemiluminescent detection kit (Thermo Scientific) based on

manufacturer’s instructions using NF-κB (5’-Biotin-AAGTTGAGGGGACTTTCCCAGGCT-3’

and 5’-Biotin-AGCCTGGGAAAGTCCCCTCAACTT-3’) oligonucleotides. Oct1 (5’-Biotin-

TGTCGAATGCAAATCACTAGAA-3’ and 5’-Biotin-TTCTAGTGATTTGCATTCGACA-3’)

was used as the loading control.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed on nuclear cell lysates using the

SimpleChIP Enyzmatic Chromatin IP Kit (Cell Signaling Technology) based on manufacturer’s

instructions. The promoter primer sequence used for IKKα was: (sense) 5’-

GTGGTTCCGTTCAGCCCT-3’, (antisense) 5’-TGCTCGCGCGTCTTTG-3’.

Statistical analysis

Differences in the migratory and invasive ability, average tumor area and osteolytic area,

and protein expression between experimental conditions were compared by unpaired Student’s t-

test. Differences in the mRNA expression of pro-metastatic genes in the shCITED2-expressing

cells relative to scramble cells normalized to 1.0, was compared by one sample t-test. CITED2

mRNA expression in tissues and the results of the invasion assay upon IKKα re-expression were




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analyzed by ANOVA and Tukey’s multiple comparison test. p-values below 0.05 were

considered statistically significant. For all figures, (*) denotes p < 0.05, (**) denotes p < 0.01

and (***) denotes p < 0.001.

Results

CITED2 expression is elevated in breast cancer metastasis

Previously, we presented evidence that CITED2 expression is significantly elevated in

primary human breast tumors relative to normal mammary epithelium, and is negatively

correlated with survival (11, 15). Extending our analysis to metastatic lesions, CITED2 mRNA

expression was significantly higher in human breast cancer metastases relative to primary breast

tumors by qRT-PCR analysis (Fig. 1A). This difference appeared to be due to the fact that

CITED2 levels in metastases were more frequently elevated beyond those observed in normal

mammary epithelium as compared to primary tumors, many of which displayed CITED2 levels

equivalent to that in normal. Consistent with mRNA results, this expression pattern was also

appreciated at the protein level in a limited subset of samples by immunohistochemical analysis

(Fig. 1B). Lastly, among metastases, higher expression of CITED2 mRNA was observed in

metastasis to bone relative to non-bone sites. Taken together, these data demonstrate that

CITED2 expression is frequently elevated in metastatic lesions of breast cancer patients, with

highest levels in bone metastasis.

Down-regulation of CITED2 inhibits breast cancer metastasis

To explore the role of CITED2 in breast cancer metastasis, we utilized the human breast

cancer cell lines MDA-MB-231 and MDA-MB-468. These cell lines are highly invasive in vitro,




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readily establish metastases following systemic administration in animal models (18-22), and as

we have shown previously, express elevated levels of CITED2 relative to human mammary

epithelial cells and breast cancer cell lines that are non-metastatic in animal models (15). MDA-

MB-231 and MDA-MB-468 cells were stably infected with a lentiviral expression vector

containing either shRNA specific for CITED2 (shCITED2) or scrambled shRNA (scramble) and

levels of CITED2 were assessed at both the mRNA and protein levels (Fig. 2A). Stable

expression of shCITED2 resulted in a greater than 75% reduction in CITED2 expression in both

MDA-MB-231 and MDA-MB-468 cells by qRT-PCR and Western analysis. Prior to examining

the effect of CITED2 on metastatic progression, we first examined whether reducing CITED2

expression altered the rate of cell proliferation in vitro. As determined by MTS assay,

shCITED2 cells exhibited a similar growth rate to that of scramble cells, indicating that

knockdown of CITED2 did not affect cell growth in either cell line (Fig. 2B). To begin exploring

CITED2 involvement in metastatic progression, we evaluated the effects of CITED2 knockdown

on the migratory and invasive ability of MDA-MB-231 and MDA-MB-468 cells using in vitro

trans-well migration and invasion assays, respectively. CITED2 knockdown significantly

reduced the migratory and invasive ability of both MDA-MB-231 and MDA-MB-468 cells (Fig.

2C and D). Next, we assessed the effects of CITED2 down-regulation on the establishment of

metastasis following intra-cardiac administration of MDA-MB-231 and MDA-MB-468 cells

stably expressing shCITED2 or scramble in athymic nude mice. While this model does not

replicate the entire metastatic cascade, it effectively assesses the ability of tumor cells to

extravasate from the vasculature, invade and colonize secondary organ sites, and establish

vascularized metastatic lesions. Mice administered with shCITED2-expressing cells displayed

significantly reduced metastasis to bone relative to the scramble group. As evidenced by digital




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radiography, osteolytic area was significantly lower in the shCITED2 group relative to the

scramble group (Fig. 2E and F, top). Consistent with reduced osteolysis, the shCITED2 group

also displayed a significant reduction in tumor area relative to the scramble group, as measured

on histological sections (Fig. 2E and F, bottom). Additionally, mice injected with shCITED2-

expressing MDA-MB-231 cells developed fewer brain metastases relative to those injected with

scramble cells, although no differences were observed using MDA-MB-468 cells

(Supplementary Fig. S1A). Further, metastasis to the lung and liver did not appear to be affected

by shCITED2 expression in either MDA-MB-231 or MDA-MB-468 cells (Supplementary Fig.

S1B and S1C). Taken together, these observations indicate that CITED2 may play a role in the

establishment of breast cancer metastasis, particularly to the bone.

Inhibition of CITED2 reduces expression of IKKα and pro-metastatic NF-κB target genes

To explore the mechanism through which CITED2 influences metastatic ability, we

examined the effect of CITED2 knockdown on gene expression in MDA-MB-231 cells by

cDNA microarray analysis (Supplementary Table 1). Notably, IKKα, a critical mediator of the

NF-κB signaling cascade (23) was found to be down-regulated along with several downstream

targets having reported roles in promoting metastasis, including osteopontin (OPN), matrix

metalloproteinase 9 (MMP9), urokinase type plasminogen activator (uPA), secreted protein,

acidic, cysteine-rich (SPARC), interleukin-11 (IL-11) and interleukin-1β (IL-1β). While the

extracellular matrix protein SPARC and proteases MMP9 and uPA have been shown to promote

tumor invasion (24-28), evidence suggests that the cytokines IL-11 and IL-1β facilitate the

establishment of osteolytic metastasis (18, 29). Additionally, the multi-functional protein OPN

reportedly affects numerous steps in the metastatic cascade (30). Regulation of these genes by




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NF-κB signaling was confirmed in MDA-MB-231 cells, wherein treatment with the IKK

inhibitor PS1145, which prevents IκBα degradation (31), reduced both basal NF-κB signaling by

Western analysis and expression of OPN, MMP9, uPA, SPARC, IL-11 and IL-1β by qRT-PCR

(Supplementary Fig. S2A and S2B). Moreover, using qRT-PCR and Western/ELISA analyses,

we confirmed down-regulation of IKKα along with the aforementioned NF-κB target genes in

shCITED2-expressing MDA-MB-231 and MDA-MB-468 cells compared to scramble cells at

both the mRNA and protein levels (Fig. 3A-F). Further, by ChIP assay in MDA-MB-231 cells,

CITED2 was found to localize to the promoter of IKKα indicating a potentially direct role for

CITED2 in the regulation of its expression. (Fig. 3G). Collectively, these data indicate that

CITED2 knockdown reduces the expression of IKKα and several downstream pro-metastatic

genes in breast cancer cells.

Down-regulation of CITED2 attenuates NF-κB signaling

NF-κB signaling is constitutively active in breast cancer (32) and occurs via both

canonical and non-canonical pathways, each of which involves IKKα. In the canonical pathway

the trimeric IKKα/β/γ complex phosphorylates the NF-κB inhibitor IκBα, inducing its

degradation and concomitantly releasing p65/p50 transcription factors to translocate into the

nucleus for regulation of gene expression (33). In the non-canonical pathway, phosphorylation of

p100 by IKKα triggers nuclear translocation of the RelB/p52 transcription factors for regulating

gene expression (33). Since CITED2 knockdown resulted in the down-regulation of IKKα

expression along with several downstream targets of NF-κB, we next investigated the effect of

CITED2 on basal NF-κB signaling in breast cancer cells. Examining the effects of CITED2

knockdown on the expression levels and localization of NF-κB signaling factors by Western




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analysis revealed increased levels of IκBα and reduced nuclear levels of p65 and RelB in

shCITED2-expressing MDA-MB-231 and MDA-MB-468 cells relative to scramble cells (Fig.

4A), indicating that both the canonical and non-canonical pathways were affected. Consistent

with reduced nuclear p65 and RelB levels, NF-κB DNA binding activity of the p65/p50

heterodimer was also markedly reduced in shCITED2-expressing MDA-MB-231 and MDA-MB-

468 cells relative to scramble cells as determined by EMSA (Fig. 4B). Together, these data

demonstrate the ability of CITED2 to influence NF-κB signaling.

Restoration of IKKα expression reverses effects of CITED2 knockdown on breast cancer

cell invasion

Since CITED2 knockdown reduced levels of IKKα along with numerous downstream

factors with reported roles in promoting metastatic dissemination (Fig. 3), we next investigated

the possibility that the reduced metastatic ability of shCITED2-expressing breast cancer cells

was related to the down-regulation of IKKα expression. To address this question, we transiently

restored IKKα expression in shCITED2-expressing MDA-MB-231 and MDA-MB-468 cells

(Fig. 5A) and examined invasive ability by in vitro trans-well invasion assay. Notably, restoring

IKKα expression in shCITED2-expressing cells significantly increased invasive ability relative

to empty vector-transfected shCITED2 cells, returning invasiveness to levels commensurate with

those observed in scramble cells (Fig. 5B and C). These data support a role for IKKα in

mediating the effects of CITED2 on the metastatic ability of breast cancer cells.

Discussion




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Despite current treatment efforts, the vast majority of patients diagnosed with metastatic

breast cancer ultimately succumb to this disease, highlighting the need for clearer understanding

of the drivers of metastasis and their mechanism of action. In this study, we have shown that

expression of the non-DNA binding transcriptional co-activator CITED2 is significantly elevated

in metastatic lesions of breast cancer patients relative to primary tumors. Further, we have

demonstrated that down-regulation of CITED2 significantly attenuates invasive and metastatic

ability in two human breast cancer cell lines (MDA-MB-231 and MDA-MB-468). Lastly, we

provide evidence that the effects of CITED2 on metastatic ability are mediated, at least in part,

by controlling expression of the NF-κB regulator IKKα, the levels of which have been shown to

negatively correlate with relapse-free survival in breast cancer patients (Supplementary Fig. S3).

While elevated levels of CITED2 were found in patient samples of metastatic disease

relative to primary tumors, highest levels were noted in metastases to bone (Fig. 1A). Further,

the inhibition of metastatic colonization observed following knockdown of CITED2 in breast

cancer cell lines (Fig. 2E and F; Supplementary Fig. S1) was largely limited to skeletal disease.

These findings are in agreement with our previous data demonstrating that reducing CITED2

expression in the murine mammary tumor cell line NT2.5 significantly reduces bone metastasis

in vivo (15), highlighting the potential importance of CITED2 as a critical mediator of bone

metastasis in breast cancer. Although the mechanism by which CITED2 mediates this effect

remains unclear, CITED2 knockdown in MDA-MB-231 and MDA-MB-468 cells resulted in

reduced expression of IL-11 and IL-1β, both of which are reported mediators of bone metastasis

and osteolysis (18, 29), thus warranting further investigation into their potential contribution

towards the metastatic effects of CITED2.




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In addition to the impairment of bone metastasis, CITED2 knockdown in MDA-MB-231

cells also inhibited the establishment of brain metastasis. This effect was not observed in MDA-

MB-468 cells, possibly due to the fact that this cell line colonized the brain less frequently than

MDA-MB-231 in the control condition. While the lesser ability of MDA-MB-468 cells to

colonize the brain could be due to various differences between these two cell lines, it is

interesting to note that canonical TGF-β signaling is absent in MDA-MB-468 cells due to the

lack of Smad4 in this cell line (34). Combined with the reported role of CITED2 in regulating

TGF-β signaling through Smad interactions (9), it is tempting to speculate that the divergent

ability of MDA-MB-231 and MDA-MB-468 cells to establish brain metastasis may be related to

TGF-β responsiveness. Moreover, it should be noted that the lack of canonical TGF-β signaling

in MDA-MB-468 cells did not impact the effect of CITED2 knockdown on invasion or the

establishment of bone metastasis. This indicates that these effects were mediated in a TGF-β-

independent manner, further supporting a role for NF-κB signaling, which was attenuated in both

the MDA-MB-231 and MDA-MB-468 cell lines.

Despite the ability of CITED2 to directly regulate expression of the NF-κB pathway

regulator IKKα, it is not yet clear how CITED2 modulates NF-κB signaling and transcriptional

activity in breast cancer cells. Although we did not observe changes in the mRNA expression of

NF-κB signaling intermediates downstream of IKKα, upstream mediators also exist whose

expression could be impacted by CITED2. Additionally, CITED2 is known to interact with

CREB-binding protein (CBP) and p300, reported co-activators of p65-mediated gene

transcription (35), suggesting that CITED2 could also regulate the activity of NF-κB as part of

the transcriptional complex. Although the ability of IKKα to restore NF-κB signaling (data not

shown) and rescue tumor invasion in MDA-MB-231 and MDA-MB-468 cells following CITED2




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knockdown (Fig. 5B and C) implicates involvement of the NF-κB pathway, it should be noted

that IKKα can also exert NF-κB-independent effects (36). Thus, further investigation is required

not only to assess the mechanism whereby CITED2 modulates NF-κB activity, but also to

determine the extent of its contribution to the pro-metastatic effects of CITED2, as well as the

ultimate effectors of its action. Addressing these questions will not only further our

understanding of CITED2 action in breast cancer, but may also provide new avenues for the

prevention and treatment of metastatic spread.

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Figure legends

Figure 1.

CITED2 expression is elevated in human breast cancer metastasis. A, CITED2 mRNA

expression was determined by qRT-PCR in human normal mammary epithelium (n=12), primary

breast tumor tissues (invasive ductal carcinoma) from patients surviving greater than (n=11) and

less than (n=8) five years from the time of diagnosis, and metastatic lesions obtained from non-

bone (n=19) and bone (n=6) sites. (p* < 0.05; p** < 0.01). B, Immunohistochemical analysis




23

was performed on paraffin-embedded tissue sections of normal mammary epithelium (n = 5),

invasive ductal carcinoma (IDC) (n = 5) and breast cancer bone metastasis (BBM) (n = 8) using

goat anti-CITED2 antibody. Protein was visualized using DAB (brown). Sections were

counterstained with hematoxylin and visualized by light microscopy. N: Normal, T: Tumor and

B: Bone. Magnification: 400X.

Figure 2.

CITED2 knockdown in breast cancer cells inhibits tumor migration and invasion in vitro and

bone metastasis in vivo. A, CITED2 mRNA expression (left) was determined by qRT-PCR. Data

are representative of triplicate experiments. CITED2 protein expression (right) was determined

by Western analysis performed on equal amounts of protein from total cell lysates. GAPDH

serves as the loading control. B, Cell proliferation was determined by MTS assay. C and D, The

migratory and invasive capability of the tumors was determined by migration (C) and invasion

(D) assays, respectively. E and F, scramble or shCITED2-expressing MDA-MB-231 (E) and

MDA-MB-468 (F) cells were injected into the left cardiac ventricle of athymic nude mice. Top:

Digital radiographic imaging of the femur and tibia showing areas of osteolysis marked by white

circles. The adjacent histograms represent the average osteolytic area measured on the

radiographic images between the experimental groups. Bottom: H&E staining of decalcified bone

sections indicating presence of tumor lesions as shown by black rectangles and circles. The

adjacent histograms represent the average tumor area measured on histological images between

the experimental groups. (p* <0.05, p** < 0.01, p*** < 0.001).

Figure 3.




24

CITED2 knockdown in breast cancer cells reduces expression of IKKα and pro-metastatic NF-

κB target genes. A and D, mRNA expression was determined by qRT-PCR. Data are

representative of triplicate experiments. B and E, Protein expression of IKKα was determined by

Western analysis of total cell lysates (CE). GAPDH serves as the loading control. Protein

expression of MMP9 and uPA was determined by Western analysis of serum free condition

medium (CM). C and F, protein expression of IL-11 and OPN (MDA-MB-231 only) was

analyzed by ELISA. G, Localization of CITED2 or IgG to the IKKα promoter was assessed by

ChIP assay using anti-sheep CITED2 or sheep IgG antibodies in wild-type MDA-MB-231 cells.

(p* <0.05, p** < 0.01, p*** < 0.001).

Figure 4.

CITED2 regulates NF-κB signaling. A, Western analysis of cytoplasmic IκBα and nuclear p65

and RelB proteins was performed on equal amounts of protein obtained from cytoplasmic (CE)

and nuclear (NE) cell lysates. GAPDH and HDAC1 serve as the cytoplasmic and nuclear loading

controls, respectively. B, Non-radioactive EMSA analysis was performed on equal amounts of

nuclear cell lysate using NF-κB and Oct-1 oligonucleotides. Oct-1 serves as the loading control.

(p* <0.05, p** < 0.01).

Figure 5.

Restoration of IKKα expression following CITED2 knockdown in breast cancer cells rescues

cell invasiveness. A, IKKα expression was determined by Western analysis of equal amounts of

total protein lysates obtained from scramble cells or shCITED2-expressing cells transfected with

either empty vector (EV) or IKKα. GAPDH serves as the loading control. B and C, Invasive




25

ability of scramble cells and shCITED2 cells transfected with either empty vector (EV) or IKKα

was determined by in vitro trans-well invasion assay. (B) Representative images of trans-well

inserts following staining of invading cells with crystal violet (purple). (C) Quantification of

invading cells showing the average number per experimental condition. (p*** < 0.001).




Figure 1.

A

B

0

250

500

750

1000

Primary tumors

*

1000

3000

5000

Metastases

**

Normal Mammary

Epithelium

> 5 yr

Survival

< 5 yr

SurvivalNon-Bone Bone

*R

ela

tive m

RN

A E

xp

ressio

n

Mammary epithelium IDC (high CITED2) IDC (low CITED2) BBM

N

T

T T

T

T

N B

T

N

N

T

T




0 24 48 72 960.0

0.5

1.0

1.5

2.0 scrambleshCITED2

Hours

MTS

Assay (

OD

)

scra

mble

shCIT

ED2

0

250

500

750

1000

1250

1500

1750

*

Num

ber

of m

igra

ting c

ells

scra

mble

shCIT

ED2

0

100

200

300

400

500

*

scra

mble

shCIT

ED2

0

400

800

1200

1600

***

Num

ber

of in

vadin

g c

ells

scra

mble

shCIT

ED2

0

1000

2000

3000

*

C D

Figure 2.

A B

231 468

231 468

231 468

scra

mble

shCIT

ED2

0.0

0.5

1.0

1.5

*

Ave

rag

e o

ste

oly

tic a

rea

(m

m2)

scra

mble

shCIT

ED2

0.0

0.5

1.0

1.5

2.0

2.5

*

Ave

rag

e t

um

or

are

a (

mm

2)

E F scramble shCITED2

2

31

scramble shCITED2

4

68

scra

mble

shCIT

ED2

0.0

0.5

1.0

1.5

2.0

2.5

*

Ave

rag

e t

um

or

are

a (

mm

2)

scra

mble

shCIT

ED2

0.0

0.1

0.2

0.3

0.4

0.5

*A

ve

rag

e o

ste

oly

tic a

rea

(m

m2)

shCITED2

0 24 48 72 960.00

0.25

0.50

0.75

1.00 scramble

Hoursscra

mble

shCIT

ED2

0

25

50

75

100

**

Re

lativ

e m

RN

A e

xpre

ssio

n

231 468

CITED2

GAPDH

CITED2

GAPDH

23

1 4

68

scra

mble

shCIT

ED2

0

25

50

75

100

**




IKK

MM

P9uP

A

SPARC

IL11

IL1

-100

-50

-7.5

-5.0

-2.5

0.0

*

** ***

*

*

Rela

tive m

RN

A e

xpre

ssio

n2

31

46

8

Figure 3.

A B C

D E F

MMP9

uPA

CE

C

M

IKKα

GAPDH

MMP9

CM

uPA

IKKα

CE

GAPDH

scra

mble

shCIT

ED2

0.0

0.1

0.2

0.3

0.4

0.5

*

OP

N e

xpre

ssi

on (

ng/m

l)

scra

mble

shCIT

ED2

0

10000

20000

***

30000

40000

50000

IL11 e

xpre

ssio

n (

pg/m

l)

scra

mble

shCIT

ED2

0

10000

15000

17500

20000

22500

25000

**

IL11 e

xpre

ssio

n (

pg/m

l)

G

23

1

IgG

CIT

ED2

0.00

0.05

0.10

0.15

0.20

0.25

*

% I

nput

IKK

OPN

MM

P9

uPA

SPAR

CIL

11IL

1-25

-15

-5-5.0

-2.5

0.0

***

**

**

*

*

Rela

tive m

RN

A e

xpre

ssio

n




Figure 4.

IkBα

GAPDH

CE

p65

RelB

HDAC1

NE

231 468 A

B

46

8 2

31

NF

-B

Oct1

p65.p50

p50.p50

p65.p50

p50.p50

Oct1

NF

-B




A

231 468

IKKα

GAPDH

Figure 5.

IKKα

GAPDH

B

scra

mbl

e

shCIT

ED2

(EV) )

shCIT

ED2

(IKK

0

200

400

600

800 ***

***

Num

ber

of

inva

din

g c

ells

scra

mbl

e

shCIT

ED2

(EV) )

shCIT

ED2

(IKK

0

100

200

300

400***

***

Num

ber

of

inva

din

g c

ells

scramble

shCITED2

(EV)

shCITED2

(IKKα)

scramble

shCITED2

(EV)

shCITED2

(IKKα)

C




Published OnlineFirst May 23, 2016.Mol Cancer Res Swaathi Jayaraman, Michele Doucet, Wen Min Lau, et al. Effects on IKKalphaCITED2 Modulates Breast Cancer Metastatic Ability Through

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