Download Supplementary methods, legends and Table S1

Supplementary Materials and Methods:
Lentivirus transfection, siRNA and cell proliferation assays: Lentiviruses were
produced in 293T cells and breast cancer cell lines infected with lentiviruses were
selected using puromycin. siRNA against AKT1 and AKT2 was obtained from Qiagen
(Valencia, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Lipofectamine (Life Technologies, Carlsbad, CA, USA) was used for transfecting siRNA
and western blotting was done four days after siRNA transfection. MCF-7 and BT-474
cells were maintained in MEM plus 10% fetal calf serum and switched to phenol red-free
MEM with 5% charcoal stripped fetal calf serum four days prior to experiments. Cell
proliferation assays were performed using Bromodeoxyuridine incorporation-ELISA
(Calbiochem/Millipore, San Diego, CA, USA). PI3K/mTOR/AKT inhibitors were
purchased from Selleck Chemicals (Boston, MA, USA).
Microarray data analyses: Genes that showed statistically insignificant signals in
majority of samples were removed from the microarray data, and only those probes that
showed statistically significant signal in at least half of the samples of at least one group
were retained. The probe level data was then collapsed to gene level data by retaining
only the probes, which showed a maximal coefficient of variation across all samples. The
data was imported into partek genomics suite for differential expression analysis.
ANOVA analysis was performed to identify genes differentially expressed between
AKT1KD vs. pLKO, AKT2KD vs. pLKO, and AKT1KD vs. AKT2KD groups. Genes
differentially expressed at p value of <0.01 were considered further for pathway analysis
using Ingenuity Pathway analysis software (© Ingenuity systems, CA, USA).
Description of supplementary files: The following additional data are available in
the online version of the manuscript.
Table S1: Sequences of primers used in qRT-PCR
Table S2: Genes differentially expressed in pLKO (labeled CTR), AKT1KD (labeled
AKT1), and AKT2KD (labeled AKT2) with or without E2 treatment for three hours.
ER binding pattern to these genes is also indicated. UNTR- vehicle treated controls
Table S3: AKT1 and AKT2-specific E2-regulated genes. Basal expression was
normalized between cell lines before calculating fold changes in E2-treated condition.
Binding pattern of ER, FOXA1, GATA3, p300, CBP, and SRC1,2,3 to these genes is
also shown.
Table S4: AKT1-AKT2_FOXA1_Comparison gene: FOXA1:E2 dependent genes are
preferentially associated with AKT1. The effects of AKT1 or AKT2 knockdown on the
expression of 83 FOXA1-dependent E2-regulated genes were examined. Eight of these
genes were found to be associated uniquely with AKT1 (Red). E2F Signature: The effect
of AKT1 and AKT2 knockdown on the expression of ER:E2F dependent genes
associated with resistance to aromatase and PI3K/mTOR inhibitors. Both isoforms had
similar effect on the expression of these genes.
Figure S1: Ingenuity pathway analysis of AKT1-dependent E2-regulated genes. Three
major pathways are shown.
Figure S2: Ingenuity pathway analysis of AKT2-dependent E2-regulated genes. Three
major pathways are shown.
Figure S3: Prognostic value of E2:ER:FOXA1:AKT1 signature in independent breast
cancer datasets. Kaplan-Meier curves for recurrence-free or overall survival of patients
with breast cancer subclassified based on ER and Progesterone Receptor (PR) expression
or tamoxifen treatment. Tumors were split at median to classify as high or low
expressers.
Table S1: Primers used for qRT-PCR
Forward
BTG2
TTC CCA GAC CTG CTT
CCA GTC TTT
KCNK6
TGG AGA ACT GCC CTT
ATG GAG CTT
RERG
TGC ACT GGA GAA
GGG AAC ATC ACA
RET
TCA CAG ATG CAC AAC
ACT CCT CCA
SIAH2
GCC TGT TGG TGA TTT
GGA TGC TGT
SLC22A5
TGC TGT GGA AAC CTC
CTT GCT ACT
SALL4
TGG AAG GAA GTT
GGC CAT CGA GAA
BETA-ACTIN
AAT GRG GCC GAG
GAC TTT GAT TGC
Reverse
ACA AGA TGC AAG
AAC ACA CAG CCT GC
TGT GCT TTC AAC ACC
TCA CCT CCT
TAA TGG CTT GCT TGA
CAT GCG TGG
TAC AGT GCT GAC AAC
ACA GCC AGA
TCA AGG GAC CAA TAT
GGG AAG GCA
AAC CAG CCT TTC CCA
AGT GCA TTC
TGT ACT GGT TCC ACA
CAA CAG GGT
AGG ATG GCA AGG
GAC TTC CTG TAA