Article title: PI3K independent activation of mTORC1 as a target in
lapatinib-resistant ERBB2+ breast cancer cells
Journal: Breast Cancer Research and Treatment
Authors: Anna-Maria Jegg1, Toby M. Ward1, Elizabeth Iorns3, Nicholas Hoe4,
JinYao Zhou4, Xiaofei Liu1, Sharat Singh4, Ralf Landgraf2 and Mark D. Pegram1
1Division
of Hematology and Oncology, University of Miami Sylvester
Comprehensive Cancer Center, Miami, FL 33136
2Department
of Biochemistry and Molecular Biology, University of Miami Miller
School of Medicine, Miami, FL 33136
3Science
Exchange, Inc. Palo Alto, CA 94301
4Prometheus
Laboratories, San Diego, CA 92121
Corresponding author: Mark D. Pegram; mpegram@stanford.edu
Quantitative real time PCR (qPCR):
RNA was isolated from SKBR3 and SK-lapR cells with Trizol (Invitrogen) and
cDNA was synthesized using SuperScript III First Strand Synthesis System for
RT-PCR (Invitrogen) with oligo(dT) according to the manufacturer’s instructions.
Real-Time qPCR was performed using SYBR Green I Master mix (Roche
Applied
Science)
and
EGFR
GCACCTACGGATGCACTGGGC-3’
specific
primers
and
(EGFR_Fwd:
EGFR_Rev:
5’5’-
AACGATGTGGCGCCTTCGCA-3’) on the LightCycler® 480 (Roche Diagnostics)
according to the manufacturer’s instructions. Standard curves were calculated for
all reactions with serial dilutions of SKBR3 cDNA to determine reaction efficiency.
The expression of EGFR-mRNA is shown relative to expression in SKBR3 cells
after normalization to GAPDH as endogenous control. [mean ± SD of triplicate
experiments]
Fluorescent in situ hybridization (FISH) analysis
Preparation of formalin fixed and paraffin embedded cell pellets:
SKBR3 and SK-lapR cells (1-5 x 107) were collected by trypsinization, washed
twice with 1xPBS and fixed in 10ml of a 10% (v/v) neutral-buffered formalin
solution over night. The following day cells were pelleted by centrifugation at 500
x g for 10 minutes, formalin solution was removed and cells were resuspended in
3-4ml of liquid 3% low melting point (LMP) agarose. Before solidification, aliquots
of 1.2 ml were transferred to 1.5ml microcentrifuge tubes and cells were pelleted
by quick spin and incubated at 4°C for 15 minutes. The agarose embedded cell
pellet for each cell line was carefully removed from the microcentifuge tube and
transferred to an embedding cassette, which was then placed in a 70% ethanol
solution for 24 hours before the cell pellet was embedded into paraffin blocks.
EGFR-FISH analysis:
Sectioning of cell pellets embedded in paraffin blocks and subsequent FISH
analysis with EGFR specific DNA probes (LSI EGFR 7p12 amplification probe)
was performed at the cytogenetic and molecular diagnostic laboratory at the
University of Miami Miller School of Medicine. A probe for chromosome 7
centromere (CEP7, D7Z1) was used as control and to determine gene
amplification via the ratio of EGFR/CEP7. FISH signals for EGFR and
centromere 7 were scored in a total of 200 cells per cell line using the criteria
described in (1).
1.
Varella-Garcia M. Stratification of non-small cell lung cancer patients for
therapy with epidermal growth factor receptor inhibitors: the EGFR fluorescence
in situ hybridization assay. Diagnostic pathology. 2006;1:19.