High basal Wnt signaling is further induced by PI3K/mTor inhibition but sensitive to cSRC inhibition in mammary carcinoma cell lines with HER2/3 overexpression

Canine mammary cell lines and culture

Canine mammary tumor cell lines used in this study were CMT1, CMT-U229, CMT-U335,
CMT-U27, CMT9, P114, CHMp, CHMm, CNMp, CNMm, CIPp and CIPm 19]–21]. The cell lines were generous gifts of the Prof Dr Hellmen (SLU, Uppsala, Sweden),
Prof Dr Sasaki (Laboratory of Veterinary Surgery, University of Tokyo, Japan), and
Dr Rutteman (Utrecht University, The Netherlands). All cell lines were cultured in
DMEM/F12 (Invitrogen, Bleiswijk, The Netherlands) supplemented with 10 % fetal bovine
serum (FBS) (FBS Gold, PAA, C?lbe, Germany). Cells were tested to be free from mycoplasma
with a Mycosensor QPCR assay according to manufacturer’s protocol (Agilent technologies,
Middelburg, The Netherlands).

TCF-reporter assay

Cells were seeded in a 24 well plate (Primaria, BD Biosciences, Breda, The Netherlands)
at a density of 100,000 CMT1, CMT-U27 and CMT9 cells and 80,000 CIPm cells, to reach
an 80 % density 24 h before transfection. Transfection was performed in FBS-free medium
using 3 ?l Lipofectamine 2000 (Invitrogen), 800 ng pTOPFLASH (TOP) or pFOPFLASH (FOP)
(gift from Prof Dr Hans Clevers, Hubrecht Institute, The Netherlands) and 0.5 ng human
ß-actin-promoter renilla construct 22] as an internal control. Transfection was stopped after 5 h by adding the same volume
DMEM/F12 supplemented with 20 % FBS. Cells were treated with 100 nM Everolimus (Selleckchem,
Munich, Germany), 50 nM BEZ235, (Selleckchem), 20 ?M Src-I1 (Enzo, Lausen, Zwitserland),
or 1 ?M FAK Inhibitor 14, (Santa Cruz, Heidelberg, Germany) for 40 h. All the compounds
were dissolved in DMSO and diluted in medium to a final concentration of 0.2 % DMSO.
The firefly and renilla luciferase activities were measured using a Dual-Luciferase
Assay System (Promega, Leiden, The Netherlands) in a Centro LB 960 luminometer (Berthold
Technologies, Vilvoorde, Belgium).

Real time quantitative RT-PCR

From each cell line, total RNA was isolated and treated with DNase using RNeasy mini
kit (Qiagen, Venlo, The Netherlands) according to manufacturer’s protocol. Using iScript
kit (BioRad, Veenendaal, The Netherlands), cDNA synthesis was performed. Specific
primer sets were used to amplify gene products in a qPCR reaction (Table 1). The reactions were performed and measured using a BioRad MyIQ detection system
(BioRad) with SYBR green fluorophore. Relative target gene expression was normalized
to a set of eight reference genes (tested in Genorm with a pairwise variation (PV)
of 0.07) for the transfection experiments. For the cluster analysis, 6 reference genes
were used with a PV 0.07 (HNRPH, TBP, SRPR, HMBS, RPS5 and RPS19). A relative induction
of gene expression was statistically assessed using paired, 2-tailed student’s T-test. Relative expression was calculated by the delta-delta Ct (??Ct) method 23].

Table 1. Primers used

Cell viability

Cell viability was determined by means of the colorimetric 3-[4,5-dimethylthiazol-2-yl]
2,5-diphenyltetrazolium bromide assay (MTT) (Sigma Aldrich, Zwijndrecht, The Netherlands).
Briefly, cells were seeded in 96 wells plates (Primaria, BD Biosciences, Breda, The
Netherlands) and after 24 h incubation to attach and stretch, treated with the different
compounds for 40 h. Cell viability was determined by incubating 20 ?l 5 mg/ml MTT
in 100 ?l medium in each well. After a 2 h incubation, the media was removed by decanting
and 100 ?l DMSO was added to each well, incubated for 30 min and the absorbance was
measured at 595 nm in a spectrophotometer Anthos Multimode Detector (Anthos Mikrosystem
GmbH, Krefeld, Germany). IC50 curves were plotted with Sigma-plot version 12.5.

Protein extraction and Western blot

Cells were seeded in 75 cm
²
bottles and after 24 h incubation to attach and stretch, treated with the different
compounds. After 40 h cells were washed with cold HANK’s balanced salt solution and
lysed and scraped with RIPA buffer 5]. After 20 min incubation on ice, samples were centrifuged for 15 min at 16,000 g
and 4 °C. Protein concentration was determined using Bio-Rad Dc Protein Assay (Bio-Rad
Laboratories). Twenty micrograms of protein from total cell lysates was subjected
to SDS-PAGE and analyzed by Western blot. Primary antibodies used in this study were
directed against ?-Catenin (Ab6302 1:4000) (Abcam, Cambridge, UK), HER2 (PA5-14635
1:500) (Pierce-Thermo Scientific), HER3 (PA1-86644 1:2500 (Thermo Scientific), with
?-Actin pan Ab-5 (MS-1295-P1 1:2000) (Thermo Scientific) as a reference protein. And
as secondary antibody, goat anti-mouse HRP-conjugated (HAF007), goat anti-rabbit HRP
conjugated (HAF008) and donkey anti-goat HRP conjugated (HAF109) (RD Systems, Abingdon,
UK) was used in a 1:20.000 dilution. HRP was visualized using Advance TM_Enhanced
chemiluminescence (ECL, Amersham, GE Healthcare, Eindhoven, The Netherlands) and analyzed
using GelDoc 2000 (Bio Rad). With the Quantity One software, version 4.6.9 (BioRad),
densities were measured, corrected for the background and related to ?-Actin expression
as loading control.

Statistics

Cluster analysis with qPCR results from all the cell lines was done in RStudio (version
3 software (R Core Team (2013) R: A language and environment for statistical computing.
R Foundation for Statistical Computing, Vienna, Austria; http://www.R-project.org)) with a Pearson:Spearman test (genes: Pearson; samples: Spearman average) to find
a correlation between the TOP/FOP ratio, related target genes and the cell lines.

Transfection and incubation studies, and also the MTT assays, were done in three independent
experiments (n?=?8 for MTT and n?=?4 for each transfected/incubation compound). After background subtraction, the
cell viability, the Wnt-signaling and the protein levels were calculated as a percentage
of the non-treated cells. IC50 curves were done in a single experiment with n?=?8 and the HER3 Western blots were done in triplicate. Differences in TOP/FOP activities,
RNA, cell viability and protein expression levels were statistically assessed using
unpaired, two tailed Student’s t test, a P value less than 0.01 was considered significant.