SUPPLEMENTARY METHODS
Cell lines, cell culture, and reagents
The cell line panel included 49 breast cancer cell lines and 3 immortalized breast
epithelial cell lines representing the known molecular subgroups of breast cancer as
described in detail previously[1,2]. MDA-MB-415, MDA-MB-134, HCC-1419, HCC-38,
HCC-70, HCC-1187, HCC-1806, HCC-1937, HCC-1954, MDA-MB-436, HCC-1569,
HCC-1500, ZR-75-30, HCC-202, Hs578t, HCC-1143, MDA-MB-175, BT-474, SK-BR-3,
MDA-MB-361, UACC-893, UACC-812, UACC-732, T-47D, MDA-MB-453, MDA-MB468, CAMA-1, MDA-MB-157, MCF-7, MDA-MB-435, ZR-75-1, BT-20, MDA-MB-231,
BT-549, DU-4475, HCC-1395, HCC-2218, 184A1, 184B5 and MCF-10A that were
purchased from American Type Culture Collection (Rockville, MD). The cell lines EFM192A, KPL-1, EFM-19, COLO-824 and CAL-51 were obtained from the German Tissue
Repository DSMZ (Braunschweig, Germany). Both cell line banks perform the cell lines
authentication by short tandem repeat analysis. The cell lines SUM-190 and SUM-225
were obtained from the University of Michigan (Ann Arbor, MI). Upon receipt, all cell
lines were assessed for Mycoplasma contamination using a multiplex PCR method[3]
and mitochondrial DNA from the cells was sequenced to confirm their correct identity[4].
Upon receipt, all cell lines were assessed for Mycoplasma contamination using a
multiplex PCR method[3], and mitochondrial DNA from the cells was sequenced to
confirm their correct identity[4]. Cell lines were then expanded and these procedures
were repeated for all cell lines prior to cryopreservation. All cell lines were passaged for
less than 6 months before use in this study. Trastuzumab-resistant BT-474 (BT-474-TR)
and SKBR3 (SK-BR-3-TR) cell lines (pools of resistant cells) were established after
serial passage in the continued presence of trastuzumab 105 µg/ml. Lapatinib-resistant
BT-474 (BT-474-LR) and SK-BR-3 (SK-BR-3-LR) cell lines (pools of resistant cells)
were established after serial passage in the presence of gradually increasing
concentrations of lapatinib (0.1 µM to 7 µM). A tamoxifen-resistant MCF-7 cell line was
established after a long-term estrogen deprivation (over 12 months) by serial passaging
in a RPMI medium without phenol red (Invitrogen, Carlsbad, CA) supplemented with
10% charcoal-stripped dextran treated FCS (Omega Scientific, Tarzana, CA).
4-hydroxytamoxifen (TAM) and fulvestrant (FUL; Faslodex®) were purchased
from Sigma Aldrich Co. (St. Louis, MO). Trastuzumab (Herceptin®) was purchased from
Genentech (South San Francisco, CA).
Proliferation assays
Cells were seeded in duplicate at 5 x 103 to 5 x 104 cells per well in 24-well
plates, as described previously[1]. A day after plating, cells were treated with a
concentration range of everolimus starting at 100 nM and decreasing by nine to twelve
2-fold dilutions. t = 0 control wells were counted at the time of treatment. The remaining
control and treated wells were counted after 5 days of treatment. Trypsinized cells were
placed in an Isotone solution and immediately counted using a Coulter Z1 particle
counter (Beckman Coulter Inc, Fullerton, CA). Suspension cultures were counted using
a Coulter Vi-Cell counter (Beckman Coulter Inc, Fullerton, CA).
To evaluate the combinations of everolimus with TAM and FUL, estrogen
receptor-positive (ER+) cell lines MDA-MB-415, CAMA-1, and MCF-7 were seeded in
24-well microplates as described above. Cells were treated with everolimus alone, TAM
alone, or with their combination, or everolimus alone, FUL alone, and their combination.
To evaluate the combination of everolimus with trastuzumab, HER2-amplified (HER2+)
cell lines BT-474, SK-BR-3 were seeded in microplates as described above. Cells were
treated with everolimus alone, trastuzumab alone, or with their combination. For all the
combination experiments, cells were treated in duplicate, and 2-fold dilutions over 6
fixed-ratio concentrations were performed. The cells were counted on Day 1 and Day 6
with a Coulter Z1 particle counter as described above.
For the studies with the MCF-7 tamoxifen-resistant cell line (MCF7/TAMR),
proliferation studies were performed as above except cells were plated without FBS and
were supplemented with 0.5 nM β-estradiol (Sigma). Proliferation assays were then
performed as above.
The average number of cell population doublings (or generations) from baseline
and generational percent inhibition were calculated as previously described[1]. All
experiments were performed at least twice.
Western blots
In order to determine the effect of everolimus on the expression of analyzed
proteins, cells in log-phase growth were treated with 100 nM everolimus, harvested at 6
different time points (10 min, 30 min, 1 hr, 8 hr, 24 hr, and 48 h), and lysates taken as
described previously[1]. Total AKT and S6 were detected using a rabbit polyclonal
antibody to AKT and S6 (Cell Signaling, Danvers, MA). AKT phosphorylation was
detected using a rabbit polyclonal antibody to phospho-serine 473 (Cell Signaling). S6
phosphorylation was detected using a rabbit polyclonal antibody to phospho-serine
235/236 (Cell Signaling).
Quantitative analysis of cell cycle and apoptosis by flow cytometry
The effects of everolimus on cell cycle were assessed using Nim-DAPI staining
(NPE Systems, Pembroke Pines, FL) as described previously[1]. The cells were plated
evenly in control and experimental wells, allowed to grow to log phase, and then treated
with 10 nM everolimus for 5 days. For apoptosis, cells were plated evenly in control and
experimental wells, allowed to grow to log phase, and then treated with 100 nM
everolimus for 5 days and analyzed using Annexin V-FITC and Cell Lab Quanta SC flow
cytometer (Beckman Coulter, Brea, CA) as described[1].
DNA isolation and oligonucleotide array Comparative Genomic Hybridization
(aCGH) analysis
Extraction of genomic DNA was performed from frozen cell pellets using the DNeasy
Blood and Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer’s
instructions. Genomic copy number imbalances were determined by aCGH using 105K
CGH oligonucleotide microarrays (105A, Agilent Technologies, Santa Clara, CA) as
described[5]. Data was subsequently extracted using Agilent Feature Extraction
Software and CGH Analytics software v.4.0 (Agilent Technologies) was used for copy
number analysis, employing the ADM2 algorithm (Threshold 5), with fuzzy zero and
centralization corrections to minimize background noise. All map positions were based
on the March 2006 NCBI36/hg18 genome assembly. A minimum of 3 consecutive
probes was required to define a region as amplified or deleted. Amplifications were
defined as log2 ratios greater than 1; homozygous deletions were defined as log2 ratios
less than -2. CGH analysis was performed on 48 cell lines (excluding the cell lines
conditioned for acquired resistance).
Microarray analyses of cell lines
Agilent microarray analyses were developed for each cell line. RNA extraction
was performed as described[6]. Microarrays were then performed on the Agilent Human
Whole Genome 44Kx4 chip. Characterization of individual breast cancer cell lines by
comparison to a breast cell line mixed reference pool was conducted as described[6].
Microarray slides were read using an Agilent Scanner, and the Agilent Feature
Extraction software version 9.5 was used to calculate gene expression values.
Extracted data was imported into Rosetta Resolver 7.2 to create expression profiles for
each individual breast cell line experiment. These microarray data are available with
GEO accession number GSE44552.
Statistical methods
To quantify the association between candidate genetic alterations (point
mutations, CNAs) and response to everolimus in vitro, we performed a semi-supervised
genotype-response association screen. Relative risk of response and corresponding p-
values for each predictor were determined by modified Poisson regression with robust
error variance http://aje.oxfordjournals.org/content/159/7/702.abstract using the PROC
GENMOD function in SAS for Windows version 9.2. Pearson correlation coefficients
and their corresponding p values were calculated using the PROC CORR function in
SAS.
Transcriptional expression profiles were obtained using the Agilent Human
Whole Genome 44Kx4 chip platform. The Resolver system analysis of variance
(ANOVA) and hierarchical cluster analysis of the breast cell line expression profiles
were used to compare first the sensitive cell lines (n = 13) and the resistant cell lines (n
= 25). All sequences were used that met a statistical cutoff of at least a 2.0 change in 7
experiments with a coefficient of variation >0.3.
13,539 gene probes met this
requirement. In the second ANOVA analysis, only luminal breast cell lines classified as
sensitive (n = 10) or resistant (n = 9) to everolimus were used. All sequences were used
that met a statistical cutoff of at least a 2.0 change in 4 experiments with a coefficient of
variation >0.3. 11,829 gene probes met this requirement. First, ANOVAs were
performed using the Benjamini-Hochberg False Discovery Rate (FDR) multiple test
correction. When multiple test correction was used, no probe achieved statistical
significance in both experiments. No test corrections were used in these analyses. Cell
lines classified as intermediate in sensitivity were excluded from ANOVA analyses. For
both ANOVA analyses, the criteria used to determine differentially expressed genes
were P <0.01. The two-dimensional cluster analysis was performed using an
agglomerative hierarchical clustering algorithm based on the cosine correlation similarity
metric.
All graphs and tables were created in Microsoft Excel.
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