Supplementary material and methods
Cell proliferation assay
The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTT) assay (Promega) was used to assess cell proliferation,
as described in our previous paper [1].
Western blot analysis
Cells were serum starved for 24 hours and then treated with either vehicle control
(DMSO) or various concentrations of LBH589. Cell lysates were run on a 12%
acrylamide gel, transferred to a nitrocellulose membrane, and probed with antibodies.
NF-κB1, pNF-κB1, RelA, p-RelA, and IκBα antibodies were purchased from Santa Cruz
Biotechnology. -actin, cleaved-PARP, cFLIP, p21WAF1/CIP1, Bax, Bcl-xL, AKT, ERα and
Lamin A/C antibodies were purchased from Cell Signaling Technology. Bands were
visualized by chemiluminescence using HRP-conjugated secondary antibodies.
Cell cycle analysis
Cell lines were treated with LBH589 (20 nM) or vehicle control (DMSO) for the
stated time periods. To include detached and floating apoptotic bodies, culture and wash
media were all saved and included in the analysis. Cells were washed with PBS and
detached using 0.25% trypsin-EDTA. Detached cells, culture medium, and the PBS wash
were combined and spun for 4 minutes at 200 g. To fix the cells, 1 ml of -20°C 70%
ethanol in H2O was added, drop-wise, while gently vortexing; cells were stored overnight
at 4°C. On the day of staining, cells were washed with 1% BSA in PBS and then treated
for 30 minutes with 250 μl of staining solution containing 50 μg/ml propidium iodide, 20
μg/ml RNase A, and 1% BSA in PBS. Cells were analyzed by flow cytometry using a
FACScan (Becton Dickinson) flow cytometer equipped with a blue laser (488 nm) for the
excitation source. Fluorescence emission was detected using 585/42 nm band pass filters.
FACS data were analyzed using the cell cycle calculator in the ModFit LT 3.0 software
package (Verity Software House).
Animal studies
Tumor specimens were stained using cleaved PARP antibody (Asp214, Cell
Signaling Technology) for apoptosis and Ki-67 antibody (M7240, Dakocytomation) for
cell proliferation. Antigen retrieval was done for 30 minutes in citrate buffer or EDTA
buffer at 98 oC, respectively. Immunohistochemically stained slides were quantified by
taking four random fields, counting stained and unstained cells, and dividing each by the
total number of cells counted to generate the percentage of positive cells. To measure
NF-B1 gene expression, total RNA was extracted from tumor specimens with TRIzol
reagent (Invitrogen), and then cDNA synthesized from isolated RNA and analyzed by
realtime PCR.
Real-time PCR analysis
For cell culture studies, cells were plated in 60 mm dishes at a density of 8x105
cells/dish and treated with either DMSO or various concentrations of LBH589. Twentyfour hours after treatment, cells were collected for gene expression analysis and washed
with PBS. TRIzol reagent (Invitrogen) was used for total RNA isolation. cDNA was
synthesized from isolated RNA using reverse transcriptase III (Invitrogen). Real time
PCR primers were as follows: -actin, 5’-CACCAACTGGGACGACAT-3’ and 5’GCACAGCCTGGATAGCAAC-3’; NFκB1, 5’-ATTTGAAACACTGGAAGCACGA-3’
and 5’-GCGGATTAGCTCTTTTTCCCG-3’; RelA, 5’-
GAATTCCAGTACCTGCCAGA-3’ and 5’-GGTCCGCTGAAAGGACTCTT-3’;
CFLAR (encoding cFLIP), 5’-TTTCTTTGCCTCCATCTTGG-3’ and 5’GGGGGAGTTCGTCCTGTC-3’; CCND1 (encoding Cyclin D1), 5’GCGAGGACCAGAAGTGCGAGG-3’ and 5’-AGCTTGTTCACCAGGAGCAGC-3’;
CDKN1A (encoding p21WAF1/CIP1), 5’-AGTCAGTTCCTTGTGGAGCC-3’ and 5’CATGGGTTCTGACGGACAT-3’. Reactions were run in triplicate on an iCycler iQ5
real time PCR detection system (Bio-Rad), using two-step SYBR green Supermix (BioRad), and results were analyzed with iQ5 software. The relative expression level of each
gene was normalized to the β-actin expression level for each sample.
Microarray analysis
H295R (human adenocortical carcinoma), HeLa (human cervical cancer) and
MCF-7her2 (HER2-overexpressed MCF-7) cells were treated with DMSO or LBH589
(50 nM) for 24 hours. For microarray analysis, total RNA was extracted using TRIzol
reagent (Invitrogen). Synthesis and labeling of complementary RNA (cRNA) targets,
hybridization of GeneChips, and signal detection were carried out by the Functional
Genomics Core Facility at City of Hope. The Affymetrix Human Gene 1.0 ST Array
(Affymetrix) was used for microarray gene expression profiles. The microarray was
carried out using Ambion’s WT Expression kit (Life Technologies) and Affymetrix’s
GeneChip Terminal labeling system. Briefly, 100 ng of total RNA was used to start the
first strand cDNA synthesis using an engineered random primer plus polyT7 promoter.
After the second strand cDNA synthesis, the antisense cRNA (in vitro transcription) was
generated using T7 RNA polymerase. Then 10 μg of cRNA was used to start the second
cycle of cDNA synthesis using random primers plus dUTP and dNTP. The single-strand
cDNA was fragmented and then end-labeled with biotinylated nucleotides in the presence
of terminal deoxynucleotidyl transferase (TdT) using Affymetrix’s WT Terminal
Labeling kit. Labeled single-stranded cDNA (5 µg) was hybridized with an Affymetrix
Human Gene 1.0 ST array, and the array was scanned using an Affymetrix GeneChip
Scanner 3000 7G.
Statistical processing of microarray data
Microarray samples were RMA normalized [2] using Partek® Genomics SuiteTM
(Version 6.5; Partek, Inc.), and genes were defined as differentially expressed if they
showed at least a 1.5 fold-change and false discovery rate (FDR) < 0.05 (LBH vs.
DMSO). FDR values were calculated using the method of Benjamini and Hochberg [3]
from the distribution of ANOVA with Linear Contrast p-values. Our microarray data is
MIAME compliant and the raw data has been deposited in NCBI Gene Expression
Omnibus (GEO) with accession number of GSE36509. Systems-level analysis and
statistical enrichment for groups/pathways listed in Ingenuity pathway analysis (IPA)
(Ingenuity® Systems, www.ingenuity.com) were calculated using a Fisher’s Exact test.
IPA was used to analyze genes that consistently showed up- or down-regulation
following all 3 treatments (|fold-change| > 1.5, FDR < 0.05).
Ingenuity Pathway Analysis
IPA is a Web-based software program that identifies the biological functions,
pathways, and mechanisms most relevant to a given data set of genes. Information on
individual genes is drawn from a large knowledge base of biological networks created
from millions of publications, and the networks are drawn by the Functional Analysis
feature of IPA based on the connectivity of the genes. To obtain a comprehensive view of
the differentially expressed genes, core analysis was performed, including network
generation, functional analysis and canonical pathway analysis on the filtered (|fold
change|>2, P < 0.05) genes.
Clinical samples (Immunohistochemistry)
Antigen retrieval was performed for 30 minutes in EDTA buffer at 98 oC.
Sections were incubated for 30 minutes at room temperature using antibody (1/50
dilution). Slides were washed with Dako buffer, then incubated with a polymer secondary
antibody (Envision + system; Dako), followed with the treatment of chromogen
diaminobenzidine tetrahydrochloride (DAB; Dako), counterstained with hematoxylin,
and mounted. Slides were scored by a pathologist who was blinded to the sample
information, and were evaluated by light microscopy and scored as previously described
[4]. First, a proportion ratio was assigned, which represented the estimated proportion of
positive nuclear staining tumor cells. Next, an intensity score was assigned, which
represented the average intensity of positive tumor cells (0, none; 1, weak, 2,
intermediate; and 3, strong). The proportion and intensity scores were then multiplied to
obtain a total score, which ranged from 0.6 to 2.85.
Luciferase assay
Cells were transiently transfected with the pNF-κB-luciferase expression
construct (Stratagene) using the Lipofectamine Plus reagent system (Invitrogen)
according to the manufacturer's protocol. Four hours post-transfection, cells were
incubated for 24 hours before stimulation with TNFα (10 ng/ml) for one hour. Cell
lysates were collected using passive lysis buffer and NF-κB activity assayed on a TD
20/20 luminometer (Turner Designs). Protein concentrations were assayed using the BCA
Assay (Thermo Scientific). Data is expressed as relative luciferase units/protein content.
NF-κB1 siRNA treatment
siRNA oligonucleotides against NF-κB1 and control oligonucleotides
(siControlA-A) were purchased from Santa Cruz Biotechnology. The siPORTTM
NeoFXTM transfection reagent was purchased from Ambion Inc. AI-responsive and resistant cells were seeded in 6-well plastic tissue culture dishes in C/D FBS MEM at 1 x
105 cells/well. At various time points after siRNA treatment (7.5µg), cells were harvested
for gene expression analysis.
NF-κB1 transfection
MCF-7aro cells were seeded (8 x 104 cells/well) in MEM with 10% C/D FBS in
12-well plastic tissue culture dishes. Cells were allowed to grow for 24 hours before
transfection. Cells were cotransfected with 0.01 μg/well of pCMV4 p50 plasmid
(Addgene). Mock transfection with only the Lipofectamine 2000 reagent (Invitrogen)
was used as control. To determine whether NF-κB1 over-expressing MCF-7aro acquired
AI-resistance, four hours post-transfection, cells were treated with 0, 50 or 100 nM
letrozole for 2 and 4 days., after which the MTT assay was performed and NF-κB1
mRNA expression was measured.
Animal studies (Establishment of LTET (long-term exemestane-treated) cell line
from mouse xenograft)
Briefly, one of the tumor specimens from the exemestane only-treated group was
removed, digested with trypsin to make a single cell suspension, and cultured in MEM
with Earle’s salts supplemented with 10% charcoal dextran-treated FBS, 1 mmol/l
pyruvate, 100 units/ml penicillin, 0.1 g/l streptomycin, G418 (500 μg/ml), and 1 nM
testosterone.
24 h
48 h
72 h
120 h
24 h
48 h
72 h
120 h
Exe-R
1.5
1.0
24 h
48 h
72 h
NF- B1 siRNA
**
Control siRNA
NF- B1 siRNA
**
Control siRNA
**
Control siRNA
NF- B1 siRNA
0.0
**
NF- B1 siRNA
0.5
Control siRNA
NF- B1 siRNA
**
Control siRNA
Control siRNA
NF- B1 siRNA
**
**
Control siRNA
0.0
*
NF- B1 siRNA
0.5
Relative expression
1.0
NF- B1 siRNA
NF- B1 siRNA
**
Control siRNA
Control siRNA
NF- B1 siRNA
**
**
Control siRNA
NF- B1 siRNA
0.0
**
NF- B1 siRNA
0.5
LTEDaro
1.5
Control siRNA
1.0
Relative expression
MCF-7aro
1.5
Control siRNA
Relative expression
Supplementary figures
120 h
Supplementary Fig. 1. The expression of NF-κB1 was effectively suppressed by
siRNA in all cell lines. siRNA-mediated knockdown of NF-κB1 in MCF-7aro, LTED,
and Exe-R was confirmed by real-time PCR analysis 5 days after transfection. Cells were
transfected with control siRNA or NF-κB1 siRNA. Gene expression was normalized to
-actin. Columns, mean; bars, SE. *, p < 0.05; **, p < 0.01.
Absorbance (570nm)
(ratio to control)
1.5
1.0
0.5
0.0
0
1
5
10
20
LBH589 (nM)
Supplementary Fig. 2. LBH589 is less effective in the suppression of MCF-10A, a
noncancerous breast epithelial cells. MCF10A cells were treated with the indicated
concentrations of LBH589 (LBH) or DMSO (vehicle control) for 6 days and medium was
replaced every 72 h. Cell viability was assessed by MTT assay. Five replicates were
performed for each measurement, and the mean and standard error were calculated. Data
are shown as a ratio of treated samples to untreated controls.
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