Biological reprogramming in acquired resistance
to endocrine therapy of breast cancer
H Aguilar, X Solé, N Bonifaci, J Serra Musach, A Islam, N López-Bigas,
M Méndez-Pertuz, RL Beijersbergen, C Lázaro, A Urruticoechea and MA Pujana
Supplementary Materials and methods
Cell culture and growth assays
MCF-7 cells were routinely cultured and maintained in Roswell Park Memorial Institute
(RPMI) medium containing 10% fetal bovine serum (FBS) and 2 mM glutamine.
MCF7-LTED cells were established in phenol red-free RPMI medium containing 10%
dextran-coated charcoal-stripped FBS (DCC-FBS). The DCC-FBS medium was
prepared according to methods described previously (Darbre et al., 1983). For growth
assays, MCF7 cells were depleted of steroids for three days and then seeded into 24well plates at a density of 105 cells per well in DCC-FBS medium. MCF7-LTED cells
were seeded similarly and both cultures left for 24 hours (h) prior to incubation with
different concentrations of 17βE2. Next, culture medium was changed every three days
and viability/proliferation evaluated after seven days using a standard methylthiazol
tetrazolium (MTT; Sigma-Aldrich) assay. Results are expressed as percentages relative
to vehicle-treated controls. Fulvestrant (ICI 182,780) and PD173074 were obtained
from Tocris Bioscience and Sigma-Aldrich, respectively.
Western blotting and antibodies
Cells were lysed in buffer containing 50 mM Tris-HCl pH 8, 0.5% NP-40, 100 mM
NaCl and 0.1 mM EDTA, supplemented with protease inhibitor cocktail (Roche
Molecular Biochemicals) and 1 mM NaF. Lysates were clarified twice by centrifugation
at 13,000 x g and protein concentration measured using the Bradford method (Bio-Rad).
Nuclear and cytoplasmic fractions were prepared using NE-PER Nuclear and
Cytoplasmic Extraction Reagent Kit (Pierce Biotechnology). Lysates were resolved in
SDS-PAGE electrophoresis gels, transferred to Immobilon-P (Millipore) or PVDF
membranes (Roche Molecular Biochemicals) and target proteins identified by detection
of HRP-labeled antibody complexes with chemiluminescence using ECL Western
Blotting Detection Kit (GE Healthcare). Antibodies were anti-pS473-AKT (193H12,
Cell signaling Technology), anti-CDK4 (C22, Santa Cruz Biotechnologies), antiCSNK1D (C8, Santa Cruz Biotechnologies), anti-E2F1 (KH95, Santa Cruz
Biotechnologies), anti-EGFR (1005, Santa Cruz Biotechnologies), anti-ERα (SP1,
Abcam), anti-pS118-ERα (16J4, Cell signaling Technology), anti-pS167-ERα (catalog
#2514, Cell signaling Technology), anti-pS305-ERα (Millipore), anti-ERBB2 (C18,
Santa Cruz Biotechnologies), anti-ERBB3 (11A4, Santa Cruz Biotechnologies), antiERBB4 (C18, Santa Cruz Biotechnologies), anti-FGFR2 (C17, Santa Cruz
Biotechnologies), anti-GAPDH (9484, Abcam), anti-MYC (9E10, Santa Cruz
Biotechnologies), anti-PGR (hPRa 2+hPRa 3, Thermo Scientific), anti-RAF1 (C20,
Santa Cruz Biotechnologies) and anti-TUBA (DM1A +DM1B, Abcam).
Transcriptomic analysis
RNA samples were extracted using the TRIzol Reagent (Invitrogen) and quality
evaluated in Agilent Bioanalyzer 2100. RNAs were amplified using the Ribo-SPIA
system (NuGEN Technologies) and subsequently hybridized on the microarray platform
Affymetrix U133 Plus 2.0. The quantitative expression analyses were performed using
primer sequences detailed in Supplementary Table 2, including ACTB as input control.
ESR1 expression was also evaluated using TaqMan probes (Hs00174860, Applied
Biosystems) and GAPDH as input control (Hs00266705) with similar results.
TFBS predictions
Under- and over-expressed microarray probes were converted to unique gene identifiers
in Ensembl. The remaining genes represented in the microarray were used as
background for enrichment analysis. TFBSs in promoter sequences were predicted
using the STORM algorithm (Schones et al., 2007) and position frequency matrices
used from TRANSFAC (2009.1 professional version, vertebrate matrix) (Matys et al.,
2006) and JASPAR (October 2009 version) (Bryne et al., 2008). Enrichment was
computed in GiTools (manuscript in preparation; www.gitools.org) using a binomial
distribution with P values calculated as follows:
where n is the total number of genes in the category, x is the number of differently
expressed genes in the category, and p is the frequency of differentially expressed genes
(under- or over-expressed). Multiple testing correction was performed using the
Benjamin-Hochberg approach (Benjamini and Hochberg, 1995).
Analysis of public microarray datasets
Chromatin immunoprecipitation data for E2F1-AP2, E2F4 and ERα, and ERαassociated expression data were taken from the respective references or linked
repositories (Balciunaite et al., 2005; Carroll et al., 2006; Xu et al., 2007) (GEO
GSE11324 record) and evaluated in 2x2 contingency tables using the chi-square
goodness-of-fit test or the Fisher exact probability test as appropriated. The ERα
response elements identified by chromatin immunoprecipitation assays were assigned to
a single Entrez identity based on the closest known gene locus (5’-end). Preprocessed
and normalized microarray data of breast tumor profiles and responses to letrozole and
tamoxifen were taken from the corresponding repositories (Stanford Microarray
Repository, NKI dataset, and GEO records GSE1378 and GSE5462). The GSEA
(Subramanian et al., 2005) was run using default values for all parameters, but the
median probe was used instead of the max probe as the collapse method when multiple
probe sets mapped to the same gene. Associations with breast cancer pathological (ER
tumor status) and clinical (letrozole and tamoxifen treatment responses) features were
evaluated independently.
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