JTO-D-13-00405 Revision 1
SUPPLEMENTARY INFORMATION
Contributions of KRAS and RAL in
Non-Small Cell Lung Cancer growth and progression
Sunny Guin1,2, PhD; Yuanbin Ru1,2, PhD; Murry Wynes6, PhD; Rangnath Mishra4, PhD; Xian
Lu5, MS; Charles Owens1,2, MS; Anna E. Barόn5, PhD; Vihas T. Vasu4, PhD; Fred Hirsch3,6,
MD, PhD; Jeffrey A. Kern3,4, MD; Dan Theodorescu1,2,3,*, MD, PhD
1
Department of Surgery, University of Colorado, Aurora, CO, USA
2
Departments of Pharmacology, University of Colorado, Aurora, CO, USA
3
University of Colorado Comprehensive Cancer Center, Aurora, CO, USA
4
National Jewish Health, Denver, CO, USA
5
Department of Biostatistics and Informatics, University of Colorado, Aurora, CO, USA
6
Division of Medical Oncology, University of Colorado, Aurora, CO, USA
MATERIALS AND METHODS
Quantitative Real-Time PCR (qRT-PCR) analysis
Total RNA was extracted from different cell lines (biological replicates; n=3 per cell line) by
RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Total RNA
purity and quantity were determined spectrophotometrically (A260/A280 ratio being close to 2.0 for
pure RNA). An aliquot (1.0 µg) of total RNA was reverse transcribed to obtain cDNA. Genespecific primers for human RALA were from Origene, Rockville, MD (Catalogue no. HP208542);
for
RALB
(F:
5’-
AAAAGGATCCCATCCGTCACCTTGACACTCATAGTAA-3’)
housekeeping
gene
R:
AAAAAAGCTTACGAGATGGCTGCCAACAAGAGTA-3’
(F:
were
from
5’-GGTGGCTTTTAGGATGGCAAG-3’
IDT.
R:
5’-
β-actin
5’-
ACTGGAACGGTGAAGGTGACAG-3’) was used to normalize RALA and B mRNA expression
levels. qRT-PCR was carried out with SYBR Green FastMix (Quanta Biosciences, Gaithersburg,
MD) reagent. The PCR results were analyzed using the StepOnePlus Real-Time PCR System
(Applied Biosystems, Foster City, CA). The 2−ΔΔCT method1 was used to calculate relative
changes in gene expression determined from qRT-PCR experiments. The threshold cycle, Ct,
1
which correlates inversely with the target mRNA levels, was measured as the cycle number at
which the SYBR Green emission increases above a preset threshold level.
Immunohistochemical Analysis
Patient population
Primary tumor samples were obtained from 189 consecutive NSCLC patients initially staged I–
III (Union for International Cancer Control, TNM classification of malignant tumours, 6 th edition)
who underwent curative pulmonary resection at the Medical University of Gdansk, Poland,
between 2001 and 2004. Since positron emission tomography (PET) was not available during
this period, a few patients were upstaged at surgery, thus resulting in a portion of patients with
pathological stage III (32%) and stage IV (4%), all of which were included in this study. Induction
or adjuvant chemotherapy was used in a small fraction of patients (4% and 2%, respectively).
Disease-free survival (DFS) was calculated from the date of surgery to the date of relapse
(distant or local), death of any cause or last follow-up. Overall survival (OS) was defined as the
time between surgery and death of any cause or last follow-up. Median follow-up was 5.3 years
(range: 1.1-6.9 years). Institutional Review Boards from the Medical University of Gdansk and
the University of Colorado approved this research.
Tissue microarray preparation
Tissue microarrays (TMA) were prepared as described 2. Three 1.5 mm diameter cores from
representative tumor areas were obtained from each formalin-fixed paraffin-embedded tumor
block and processed using the MaxArray service (Invitrogen, South San Francisco, CA).
Sections of 4 m were cut from TMA master blocks, mounted on slides and processed as
described below.
RALA and RALB protein expression by IHC
IHC was performed in a CLIA certified laboratory. Slides were dried at 60˚C for 1 hour and
subsequently rehydrated. Antigen retrieval consisted of 20 minutes in a Decloaking Chamber
(Biocare Medical, Concord, CA) at 105˚C using Dako Target Retrieval Solution (Dako,
Carpinteria, CA, cat#S1699) at pH 6.1 and then cooled in an ice bath. After 3 rinses with Dako
Wash Buffer the slides were placed on the Dako Autostainer. Slides were then quenched for
endogenous peroxidase using 3% H2O2 for 10 minutes then pre-incubated in Universal Blocking
Reagent (BioGenex, Fremont, CA, cat#HK085-5K) for 10 minutes to reduce nonspecific binding.
The slides were next incubated for 1 hour at room temperature with an anti-RALA mouse
2
monoclonal antibody (BD Biosciences, San Jose, CA cat#610221) at a dilution of 1:800 or an
anti-RALB mouse monoclonal antibody at a dilution of 1:400 (Abgent Inc., San Diego, CA,
cat#AT3553a). After Wash Buffer rinse, the slides were incubated with HRP labeled Dual Link
System (Dako EnVision+ cat#K4061) for 30 minutes.
Slides were rinsed again and the
chromogen was developed for 7 minutes with liquid 3, 3-Diaminobenzidine (Dako. cat#K3468).
Finally, the sections were counterstained with hematoxylin (Dako. cat#S3301), dehydrated,
cleared and coverslipped.
RALA and RALB staining was assessed on an Olympus BX41 light microscope, without clinical
information. In the evaluation of the RAL expression, the immunostained cells were graded
according to cytoplasmic and membranous staining. The cytoplasmic staining intensity within
the tumor tissue was graded on a scale of 0 (negative, no cytoplasmic staining), 1 (low, mild
cytoplasmic staining), 2 (moderate cytoplasmic staining) and 3 (strong cytoplasmic staining).
The membranous staining intensity and pattern were graded on a scale of 0 (negative, no
membrane staining), 1 (faint, partial membrane staining), 2 (weak circumferential membrane
staining), 3 (intense, circumferential membrane staining). The percentage of positive tumor cells
were calculated for each specimen, and this percentage were multiplied by the staining intensity
to obtain an H-score (range, 0-300). A “membrane plus cytoplasm” H-score was obtained by
adding cytoplasmic and membranous H-scores for each core.
Microarray Analysis
Three NSCLC patient datasets (Supp. Table 3) were used to examine whether RALA or RALB
gene expression could stratify patient overall survival or recurrence-free survival. Processed
data were obtained from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo). Patients
in each dataset were divided into two groups by two methods and were compared by Cox
proportional hazards models and log-rank tests. In the first method, patients with high (top 25%)
RALA or RALB expression at the probe set level was compared with those with low (bottom
25%) expression. In the second method, a “Risk Score” was calculated for each patient using a
leave-one-out approach by fitting a Cox proportional hazards model to the microarray data on all
other patients that included either all RALA or all RALB probe sets. The risk score was the
product of the patient’s RALA or RALB expression and coefficients given by the Cox model.
Patients with high (top 25%) risk scores were then compared to those with low (bottom 25%)
scores.
3
For KRAS and RAL genes in the univariate analysis, the probe set with the highest mean
expression across patients was selected to represent a gene’s expression if there are multiple
probe sets for the gene in a dataset. To analyze the interaction between KRAS and RAL genes,
a patient is classified as either high- or low-expressing using a gene’s median expression.
Patient groups with different expression levels of KRAS and RAL genes were compared by Cox
proportional hazards models and log-rank tests. In univariate analyses, the following
characteristics, when available, were included in Cox proportional hazards models: age, gender,
histology (adenocarcinoma vs. squamous), pathological stage (low (stages I & II) vs. high (stage
III & IV)), KRAS, RALA, and RALB expressions. P-values were given by Wald tests.
4
REFERENCES
1.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-408.
2.Dziadziuszko R, Merrick DT, Witta SE, et al. Insulin-like growth factor receptor 1 (IGF1R)
gene copy number is associated with survival in operable non-small-cell lung cancer: a
comparison between IGF1R fluorescent in situ hybridization, protein expression, and mRNA
expression. J Clin Oncol 2010;28:2174-2180.
5
SUPPLEMENTARY TABLES
Supplementary Table 1. Details of the NSCLC patient tumors used for immunohistochemical
(IHC) analysis.
Characteristics
N (%)
Age
Median
64
Range
35-85
>60
118 (62)
Gender
Males
144 (76)
Females
45 (24)
Pathological Stage
I
75 (40)
II
42 (22)
III
61 (32)
IV
8 (4)
Unknown
3 (2)
Grade
G1
20 (11)
G2
81 (43)
G3
63 (33)
Unknown
25 (13)
Histology
Squamous Cell Carcinoma
103 (54)
Adenocarcinoma
55 (29)
Large Cell
NSCLC NOS/MIXED*
Other
5 (3)
24 (13)
2 (1)
Smoking
Ever
Never
180 (95)
9 (5)
Progression-Free Survival
Years - Median
1.5
Overall Survival
Years - Median
2.1
6
*NSCLC NOS/MIXED - Non Small Cell Lung Cancer whose histology is not otherwise specified
(NOS)/ mixed adenocarcinoma and squamous cell carcinoma characteristics.
Supplementary Table 2. Multivariate analysis for RALA and B on IHC samples for overall
survival (OS).
RALA Multivariate Analysis
Parameter
Comparison
RALA IHC
<25% percentile vs.
score
>75% percentile
(Membrane)
25%-50% percentile vs.
Multivariate
95% Confidence Interval of
Multivariate
Hazard Ratio
Hazard Ratio
P-value
0.769
(0.423, 1.399)
0.3894
1.079
(0.63, 1.846)
0.7821
1.201
(0.69, 2.089)
0.5179
2 vs. 1
1.52
(0.856, 2.696)
0.1527
3 vs. 1
3.757
(2.287, 6.171)
<0.0001
4 vs. 1
7.27
(3.028, 17.456)
<0.0001
1.022
(0.999, 1.046)
0.063
0.799
(0.496, 1.286)
0.3549
1.063
(0.559, 2.022)
0.852
0.721
(0.434, 1.197)
0.2055
>75% percentile
50%-75% percentile vs.
>75% percentile
Stage
Age
Histology
Adenocarcinoma vs.
Squamous
NSCLC NOS/MIXED
vs. Squamous
Gender
Female vs. Male
RALB Multivariate Analysis
Parameter
Comparison
RALB IHC
<25% percentile vs.
score
>75% percentile
(Membrane)
25%-50% percentile vs.
Multivariate
95% Confidence Interval of
Multivariate
Hazard Ratio
Hazard Ratio
P-value
0.552
(0.314, 0.97)
0.0388
0.55
(0.313, 0.967)
0.0377
0.951
(0.555, 1.63)
0.8548
2 vs. 1
1.47
(0.84, 2.571)
0.1774
3 vs. 1
3.901
(2.365, 6.435)
<0.0001
4 vs. 1
8.603
(3.577, 20.688)
<0.0001
1.03
(1.006, 1.054)
0.0148
0.747
(0.453, 1.231)
0.2519
1.126
(0.597, 2.124)
0.7129
>75% percentile
50%-75% percentile vs.
>75% percentile
Stage
Age
Histology
Adenocarcinoma vs.
Squamous
NSCLC NOS/MIXED
vs. Squamous
7
Gender
Female vs. Male
0.73
(0.434, 1.228)
0.2355
Supplementary Table 3. Detail of NSCLC transcriptomic datasets used for bioinformatic
analysis
Dataset
GSE4716_GPL3694
GSE8894
Platform Tumors and Histology Survival *
Gender *
N=50 (30 adeno, 16
NSCLC: 15F, 35M
GPL3694
GPL570
squamous, 4 large cell)
N=138 (62 adeno, 76
squamous)
OS
Adeno: 15F, 15M
NSCLC: 34F, 104M
DFS
Adeno: 29F, 33M
N=149 (90 adeno, 35
GSE11969
GPL7015
squamous, 20 large cell, 4
adenosquamous)
OS
NSCLC: 48F, 101M
Adeno: 43F, 47M
*OS is overall survival; DFS is disease free survival l; M is male; F is female.
Supplementary Table 4. qRT-PCR analysis for RALA and B mRNA expression in non-small
cell lung cancer cell lines normalized to β-actin
RALA
Cell Lines
SW1573*
Calu-6*
H2009*
H358*
H2122*
H460*
H157*
A549*
H292
H2228
H1703
HCC4006
Calu-3
H322
2^−(delta Ct)
Average
0.014
0.013
0.012
0.013
0.012
0.020
0.014
0.006
0.007
0.014
0.009
0.015
0.014
0.006
RALB
SEM
0.0031
0.0020
0.0013
0.0020
0.0007
0.0013
0.0012
0.0001
0.0006
0.0015
0.0011
0.0018
0.0045
0.0004
2^−(delta Ct)
Average
0.00007
0.01162
0.00031
0.00049
0.00650
0.00010
0.00006
0.00014
0.00005
0.00036
0.00015
0.00025
0.00008
0.00868
SEM
0.00001
0.00102
0.00007
0.00001
0.00083
0.00001
0.00001
0.00001
0.00001
0.00001
0.00001
0.00003
0.00002
0.00080
*Cell lines with KRAS mutation
8
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. RALA membrane (Mem) and cytoplasmic (Cyto) expression do not
change as a function of NSCLC (A) stage and (B) grade as evaluated by IHC. Similarly RALB
membrane (Mem) and cytoplasmic (Cyto) expression is unchanged across NSCLC (C) stage
and (D) grade as evaluated by IHC.
Supplementary Figure 2. Overall NSCLC patient survival as a function of (A) RALA and (B)
RALB membrane expression by IHC. Overall NSCLC patient survival as a function of (C) RALA
and (D) RALB membrane + cytoplasmic expression as evaluated by IHC.
Supplementary Figure 3. Patient survival in NSCLC datasets GSE8894 and GSE11969 (Supp
Table 3) as a function of RALA (A) mRNA expression or (B) Risk Score (see Materials and
Methods). (C) Overall patient survival in NSCLC dataset GSE4716_GPL3694 as a function of
RALB expression.
Supplementary Figure 4. Densitometric analysis on the western blots in Figure 3 to study the
correlation between (A) RALA and (B) RALB protein expression and activation on the panel of
14 NSCLC cell lines. RAL expression and activation are plotted on the X and Y axis
respectively.
Supplementary Figure 5. Knockdown of RALA, RALB and RALA+RALB in NSCLC cell lines.
Cells were transfected with 100nM siRNA against RALA (siRALA), B (siRALB) or both
(siRALA+B). 72hrs after transfection cells were lysed, and detected for RAL knockdown by
western blot using specific antibodies against RALA and RALB. A luciferase siRNA transfected
cells are used as control (siCTL).
Supplementary Figure 6. RALA and B were knocked down in H2122 cells using a second set
of siRNA (siRALA II and siRALB II) using similar protocol. (A) Cells were lysed 72 hrs after
transfection and knockdown was detected by western blot. (B) H2122 cells were transfected
with RAL siRNAs: siRALA II and siRALB II and plated in agar as mentioned earlier. Colonies
formed were stained and counted as mentioned earlier. Reduced anchorage independent
9
growth was observed with RALA and B knockdown. *P<0.05 by student’s t-test. Data from one
of two experiments is shown.
Supplementary Figure 7. Western blot on the panel of NSCLC cell lines for activated and total
AKT and ERK to study signal transduction through PI3K/AKT and MAPK pathways downstream
of KRAS in these cell lines. Asterisks indicate cell lines with KRAS mutations.
10