1. 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;

2. A B C Supplementary Figure 1
SK-lapR
SKBR3
SK-lapR
2.6μM lap -
DMSO
lapatinib
AZD0530
AZD + lap
p-SRC
48h
24h 48h
24h 48h
24h 48h
24h 48h
p-SRCY416
SRC
actin
p-ERK
ERK C
SK-lapR
ERa -
DMSO
lapatinib
PD325901
PD + lap
48h
24h 48h
24h 48h
24h 48h
24h 48h
actin
p-ERK1/2T202/T204
actin
Supplementary Figure 1 Constitutively active SRC and ERK1/2 signaling does not mediate resistance to lapatinib in SK-lapR cells. a: Phospho-SRC levels are increased in SK-lapR cells, and lapatinib does not inhibit ERK1/2 phosphorylation in SK-lapR cells. SKBR3 and SK-lapR cells were treated for 24h with 2.6μM lapatinib (+) or DMSO (-) and cell lysates were analyzed by immunoblot analysis using indicated antibodies. b, c: Inhibition of SRC with 1μM AZD0530 (B) and ERK1/2 with 1μM PD (C) does not re-sensitize SK-lapR cells to lapatinib. SK-lapR cells were treated with DMSO, 2.6μM lapatinib, 1μM AZD0530 (B) and 1μM PD (C), and combinations of drugs for 4 days. Cell numbers were determined by cell count on day 0 (before treatment) and day 4. Cell proliferation is plotted as fold increase relative to the cell number on day 0 [mean ± SD of triplicate experiments]. Inhibition of SRC with AZD0530 and ERK1/2 respectively after 24h and 48h was confirmed by immunoblot analysis

3. Relative mRNA expression
Supplementary Figure 2 A B
SKBR3
SK-lapR 25
EGFR mRNA 20 15
Relative mRNA expression
[fold increase] 10 5
SKBR3
SK-lapR
EGFR/CEN ratio: 0.99 = no amplification
(346/348 in 50 cells)
EGFR/CEN ratio: = no amplification
(383/366) in 50 cells)
Supplementary Figure 2: Increased EGFR gene expression in SK-lapR cells is not mediated by EGFR gene amplification. a: Quantitative PCR (qPCR) analysis of EGFR mRNA expression in SKBR3 and SK-lapR cells. cDNAs were analyzed using primers for EGFR as well as GAPDH for normalization. Data is shown as fold increase relative to EGFR expression in SKBR3 cells. b: Representative images for EGFR- fluorescence in situ hybridization (FISH) in SKBR3 and SK-lapR cells. Formalin fixed, agarose embedded cell pellets were prepared with SKBR3 and SK-lapR cells and small sections were analyzed by FISH with EGFR and centromer 7 specific DNA probes. EGFR gene copies (red) and number of chromosome 7 centromeres (CEN; green) were counted in 200 cells/ cell line and EGFR amplification was determined by EGFR/CEN ratio. No amplification of EGFR was detected in SKBR3 or SK-lapR cells.