healthmedicinet

Primary cell isolation, cell line generation and cell culturing

Cell lines based on the human breast cancer MCF7 cell lines, which predominantly express
the IRA, IRB or IGF1R have been described previously 18]. All MCF7 derivatives were cultured in RPMI 1640 medium (Gibco, Invitrogen, Carlsbad,
CA, USA) supplemented with 10 % fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin
(Invitrogen).

Primary human mammary cells have been isolated from cryopreserved biopsies of two
individuals as described previously 21]. The two biopsies were obtained from two female patients who had undergone breast
cancer-related surgery at the Leiden University Medical Center (LUMC). Procedures
were followed according to the Dutch Medical Treatment Act and the study protocol
was compliant with “the Code of proper secondary use of human tissue in the Netherlands”
issued by the Dutch Federation of Medical Scientific Societies and approved by the
Medical Ethics committee of the LUMC (P10.226). Specimens were coded anonymously in
a way that they were not traceable back to the patient by laboratory workers. As much
as possible fat tissue was removed from the human mammary biopsies, thereafter they
were cut into 8-mm
³
pieces, which were then dried and attached to the culture flask for 30 min. Twenty
percent of FBS containing Dulbecco’s modified Eagle’s medium (DMEM)-F12 medium (Gibco/Invitrogen,
Breda, the Netherlands) was gently added and refreshed every 5 days. Around the edges
of the tissue, cells (mainly fibroblasts) started growing and after 3 weeks the culture
flask was confluent with cells. The fraction of epithelial cells was enriched by multiple
short trypsinization steps in which part of the fibroblasts were removed. For two
more passages the cells were cultured in HuMEC Ready Medium (Gibco/Invitrogen). After
this step the primary mammary cells were cultured in DMEM-F12 medium supplemented
with 10 % FBS and 100 U/mL penicillin-streptomycin (Invitrogen).

Insulin, insulin analogues and IGF1 in vitro stimulation

Prior to compound stimulation the cells were starved in 5 % charcoal/dextran-stripped
fetal bovine serum (CDFBS, GE Healthcare HyClone, Utah, USA)-containing medium. Stimulations
included: insulin neutral protamine Hagedorn (NPH) (Insuman Basal, Sanofi Aventis,
Paris, France), insulin glargine (Lantus, Sanofi Aventis), first metabolite of glargine
(M1, Sanofi Aventis), second metabolite of glargine (M2, Sanofi Aventis), glulisine
(Apidra, Sanofi Aventis), lispro (Humalog, Eli Lilly, Indianapolis, IN, USA), insulin
X10 (not marketed, Novo Nordisk, Bagsvaerd, Denmark), aspart (B28Asp, Novo Nordisk),
detemir (Levemir, Novo Nordisk) and insulin-like growth factor 1 (IGF1) (Increlex,
Ipsen, Basking Ridge, NJ, USA). All insulin analogues were dissolved in their original
vehicle solutions 18]. For the in vitro experiments 1000× stock concentrations were prepared. Except for
the first exposure experiment (Fig. 1c) in which a dose response of 10, 33 and 100 nM was used, all exposures were performed
with a concentration of 10 nM.

Fig. 1. Knockdown of signaling components critical in the INSR and IGF1R pathway reveals common
canonical core in IRA-, IRB- and IGF1R-induced proliferation signaling. a The canonical INSR and IGF1R signaling pathway with the focus on proliferative and
apoptotic biological outcomes. b Western blot analysis of the cell line panel, based on the human breast cancer cell
line MCF7 with stable retroviral overexpression (IRA, IRB and IGF1R) in combination
with a stable short hairpin knockdown (INSR and IGF1R). Cells have been treated with
0, 10, 33 or 100 nM of insulin X10 for 30 min. Downstream signaling pathway activation
of the receptors is intact as is indicated by the dose-dependent activation of p-ERK/p-AKT.
c Western blot analysis of siRNA transfection efficiency in the MCF7 IGF1R cell line,
1 day and 5 days posttransfection and the effect of the knockdown on proliferation
measured with the SRB proliferation assay. d The effect of transient knockdown of ten important signaling molecules (INSR, IGF1R,
GRB2, RAF1, ERK2, IRS1, PIK3CA, PTEN, AKT2 and GSK3B) in the INSR and IGF1R signaling
pathways on SRB proliferation measured in the different MCF7 derivatives (MCF7 IRA,
MCF7 IRB and MCF7 IGF1R). e The effect of treatment and knockdown of key signaling molecules in INSR and IGF1R
signaling on SRB proliferation measured in MCF7 IGF1R. (
^*p 0.05,
^**p 0.01,
^***p 0.001). IGF1R insulin-like growth factor 1 receptor, INSR insulin receptor, IRA A isoform of INSR, IRB B isoform of INSR; siRNA small interfering RNA, SRB sulphorhodamine B

siRNA transfection

A transient transfection method with Smartpool siRNA mix (Dharmacon Technologies,
Thermo Fisher Scientific, Lafayette, CO, USA) was used to test the effect of individual
gene on cell proliferation. For this, 10,000 MCF7 cells per well were seeded in 96-well
plates in complete growth medium. Twenty-four hours after seeding, 50 nM Smartpool
siRNA mix was delivered to the cells using a standard transfection method with DharmaFECT
4 transfection reagent (Dharmacon Technologies) according to the company’s instructions.
Twenty-four hours after transfection, the small interfering RNA (siRNA) transfection
mixture was replaced with 5 % CDFBS starvation medium for drug treatment and sulphorhodamine
B (SRB) proliferation assay.

Sulforhodamine B colorimetric assay determining cell proliferation

A SRB assay was used to measure the total amount of protein as a measure for cell
proliferation. Transfected and drug-treated cells in 96-well plates were fixed with
30 ?l 50 % trichloroacetic acid directly added to 100 ?l of assay medium per well
for 1 h at 4 °C on a shaker, washed five times with distilled water and air-dried.
Fixed cells were stained with 60 ?l of 0.4 % SRB (dissolved in 1 % acetic acid) at
room temperature on a shaker for 30 min. After the SRB protein binding, the plates
were washed five times with 1 % acetic acid to remove unbound dye and air-dried between
the washing steps. Next, the protein-bound SRB in each well was solubilized in 200 ?l
10 mM unbuffered Tris solution (pH 10) for 10 min on a plate shaker. Absorbance was
measured at 530 nm with a FLUOstar OPTIMA plate reader (BMG Labtech, Offenburg, Germany).

Western blotting

Western blotting was used to determine the knockdown efficiency of the siRNA transfection.
To prepare cell lysates for Western blot analysis, cells were washed two times with
ice-cold phosphate-buffered saline (PBS) and lysed with 1?×?SPB with 1:20 ?-mercaptoethanol.
Samples were boiled at 95 °C for 5 min and stored at ?20 °C. Before loading, samples
were denatured at 95 °C for 5 min. A total of 20 ?l (about 30 ug) protein solution
per lane was separated by SDS-polyacrlyamide gel electrophoresis on a 7.5 % acrylamide
gel and electrophoretically transferred to a polyvinylidene fluoride membrane (Merck
Millipore, Billerica, MA, USA). Prior to primary antibody probe, membranes were blocked
for 1 h at room temperature with 5 % bovine serum albumin (BSA) or 5 % milk in Tris-buffered
saline and Tween 20 (TBST) buffer (100 mM Tris, pH 7.4, 500 mM NaCl, 0.05 % Tween
20). ERK, AKT, PTEN and tubulin antibodies were probed in 1 % BSA-TBST buffer, whereas
IGF1R? antibodies were probed in 1 % milk-TBST buffer. Horseradish peroxidase (HRP)-conjugated
secondary antibody incubation was performed in 1 % BSA-TBST or 1 % milk-TBST buffer,
corresponding to the primary antibodies used. Protein bands were visualized by using
the ECL (Amersham) method, after which the membrane was scanned by using a Typhoon
9400 imager (GE Healthcare, Amersham, UK). Anti-phospho-Akt (Ser473) and anti-phospho-Erk
(Thr202, Tyr204) have been purchased from Cell Signaling Technology, Danvers, MA,
USA). For a detailed description of the methods and origin of the antibodies we refer
to our prior publications 18], 22].

Microarray studies

For the microarray, the cells were seeded at a confluence of 60 % in 6-cm plates,
starved for 2 days in 5 % CDFBS-containing medium, followed by 1 h or 6 h compound
stimulation (10 nM) in serum-free medium. Small and large RNA was isolated and purified
using NucleoSpin® miRNA isolation kit (Macherey-Nagel, Düren, Germany) according to
the manufacturer’s instructions. RNA quality and integrity were assessed by using
the Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, USA).
The Affymetrix 3? IVT Express Kit (Affymetrix, Santa Clara, CA, USA) was used to synthesize
biotin-labeled cRNA, and this was hybridized to an Affymetrix HG-U133 Plus PM Array
plate reader. Probe annotation was performed using the hgu133plus2.db package and
probe mapping was performed with the hgu133plus2cdf package installed using Bioconductor
version 3.0. Probe-wise background correction (Robust Multi-array Average expression
measure), between-array normalization (quantile normalization) and probe set summaries
(median polish algorithm) were calculated with the RMA function of the Affymetrix
package (Affy package version 1.38.1) 23]. The normalized data were statistically analyzed for differential gene expression
using a linear model with coefficients for each experimental group 24]. A contrast analysis was applied to compare each exposure with the corresponding
vehicle control. For hypothesis testing the moderated t-statistic by empirical Bayes
moderation was used followed by an implementation of the multiple testing correction
of Benjamini and Hochberg 25] using the LIMMA package 26]. The microarray data is publically available at the Gene Expression Omnibus (GEO)
database via accession number GSE65398.

RT-qPCR

For the qPCR analysis, messenger RNA from MCF7 cells (80 % confluent 6-well) or mammary
glands (30 ?g tissue) was isolated/purified using NucleoSpin® miRNA isolation kit
(Macherey-Nagel). cDNA was made using the universal cDNA synthesis kit (Exiqon, Vedbaek,
Denmark). qPCR was performed in triplicate using SYBR Green PCR (Applied Biosystems,
Carlsbad, CA, USA) on a 7900HT Fast Real-Time PCR system (Applied Biosystems). Primers
targeting the mitogenic classifiers have been manually designed and are listed in
the additional material (Table S1 in Additional file 1). qPCR data were collected and analyzed using SDS2.3 software (Applied Biosystems).
Relative gene expression was calculated after correction for ?-actin expression using
the 2
^-??Q
method. Data are presented as fold change (or log2 fold change) compared to vehicle
stimulation.

Animal experiments

Forty female 12-week-old inbred FVB/NRj mice were obtained from Janvier Labs, Orleans,
France. Housing and experiments were performed according to the Dutch guidelines for
the care and use of laboratory animals (UL-DEC-14020). RM2 food SDS (Technilab-BMI,
Someren, Holland) and water were provided ad libitum. Animals received a single subcutaneous
injection of 100 ?l compound/vehicle solution. The doses were chosen so that the glucose
drop was constant among the different compounds (see Figure S3A in Additional file
2) (glargine and insulin 100 nmol/kg, X10 1200 nmol/kg and IGF1 12.5 mg/kg). One or
six hours after the injection the mice were sacrificed, blood was collected (mini
collect, Greiner/Omnilabo, Breda, Holland), blood glucose levels were measured (Freestyle
light, 70812–70, Abbott Laboratories, Abbott Park, IL, USA), the third and fourth
mammary glands were isolated and used for Western blot protein quantification and
quantitative PCR respectively 18], 22]. For each condition (treatment/time point) four mice were included.

Statistical analysis

For the statistical analysis of the microarray data, R (version 3.1) software was
used. The rest of the analysis was performed with Graphpad Prism version 4.00 (Graphpad
Software, San Diego, CA, USA). Student’s t tests were used to determine significance between conditions. P values lower than 0.05 were considered to be significant. In all graphs the error
bars represent standard deviations.