1. Transcriptome Analysis of Spontaneous and
Induced Lung Tumors in Mice using RNA Seq
2Janaya L. Shelly| 1Carol J. Bult, PhD|
1The Jackson Laboratory, Bar Harbor, ME 04609, USA 2Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Abstract
Lung cancer is the leading cause of cancer-related deaths in the United States and is estimated to
cause 160,000 deaths in 2013. Understanding the underlying genetic basis of lung cancer is key to
identifying effective therapeutic targets and to the development of prognostic markers to guide clinical
interventions. Mouse models are a potentially powerful platform for gaining insight into the genetics of
human lung cancer. To better understand the characteristic genomic changes in mouse tumors we
used RNA SEQ to measure global transcriptional activity of two spontaneous mouse lung tumors
(adenoma and Bronchioloalveolar carcinoma) and three lung tumors (early and late stage
adenocarcinomas) from mice with targeted mutations in the Kras and Trp53 genes. RNA SEQ data
from the 5 tumors were analyzed using TopHat and Cufflinks in the Galaxy bioinformatics workflow
platform. The annotated biological functions of the 200 top expressing genes between tumor and
normal lung samples across the five tumor types were then compared. The top expressing genes in
the spontaneous adenoma were enriched in functions related to homeostatic processes and regulation
of cell death. The top expressing genes in the spontaneous bronchioloalveolar tumor were enriched in
genes related to homeostatic processes and antigen presentation. The functional categories of the top
expressing genes in the tumors with targeted gene mutations included: angiogenesis and cell motility
(in lung adenocarcinomas with lysozyme driven Cre Kras mutations), biological quality and cell motility
(in lung adenocarcinomas with a mutation in Kras), and system development and cell motility (in
adenocarcinomas with combined mutations in Kras and Trp53).
A. Mouse lung tumors used in this study
B. Methods
C. Results
VLAD results for top
expressing genes in
normal and tumor
samples for ST45
VLAD results for top
expressing genes in
normal and tumor
samples for ST31
Kras (G12D)
• Krastm4Tyj
• Adenocarcinoma
• C57BL/6J strain
• Male, 6 months
• Jackson et al. 2001. Genes and Development
15:3243 –3248
ST31
• Spontaneous Bronchioloalveolar
carcinoma
• C57BL/6J strain
• Male, 2 years, 8 months
Kras (G12D)/Trp53 null
• Krastm4Tyj/J;Trp53tm1Brn/J
• Adenocarcinoma
• C57BL/6J strain
• Male, 6 months
• Jackson et al. 2005. Cancer Research 65:10280-10288
Lyz2Cre
• Lysozyme 2 Cre
• Adenocarcinoma
• C57BL/6J strain background
• Male, 18 days
• Clausen BE, Burkhardt C, Reith W, Renkawitz R
and Forster I. Transgenic Research 1999
Aug;8(4):265-77
ST45
• Spontaneous adenoma
• C57BL/6J strain
• Male, 3 years
Normal Tumor
Normal
Normal
Normal
Normal
Tumor
Tumor
Tumor
Tumor
Acknowledgements
I would like to thank my mentor, Dr. Carol Bult and members of the Bult lab: Drs. Julie Wells, Jill Recla, and Mr. Kyle
Beauchemin, for their help with this project. Funding for this project was made possible by a JAX Cancer Center Pilot grant and
by NIH NHGRI HG007053.
The significant Biological Functions represented in top
expressing genes among the 5 tumor types and matched
normal tissues analyzed in this study are summarized
below.
Kras
ST31
Kras/Trp53
Lyz2Cre
ST45
Normal Tumor
• Antigen processing and
presentation (GO:0019882)
• Translation (GO:0006412)
• Homeostatic process
(GO:0042592)
• Regulation of apoptotic process
(GO:0042981)
• Developmental processes
(GO:0032502)
• Regulation of metabolic
processes (GO:0019222)
• Antigen processing and
presentation (GO:0019882)
• Translation (GO:0006412)
• Angiogenesis
(GO:0001525)
• Cell motility (GO:0048870)
• Developmental processes
(GO:0032502)
• Response to stress
• Biological quality
(GO:0065008)
• Cell motility (GO:0048870)
• Antigen processing and
presentation (GO:0019882)
• Cell motility (GO:0048870)
• Developmental processes
(GO:0032502)
• Homeostatic process
(GO:0042592)
• Antigen processing and
presentation (GO:0019882)
Normal Tumor
Normal Tumor
Normal Tumor
Normal Tumor
Genome wide gene expression levels in mouse lung tumors and
matched normal tissue was assessed using an RNA SEQ analysis
workflow in the Galaxy1 bioinformatics platform. The biological
processes represented by the top expressing genes was assessed
using the VLAD Gene Ontology term enrichment tool from the Mouse
Genome Informatics (MGI) database2.
A graphical depiction of the functional analysis from VLAD is shown
below. Each grey box represents a Biological Process term in the
Gene Ontology (GO).
• The yellow bar shows the fraction of high expressing genes in
normal tissue that match the GO term.
• The blue bar shows the fraction of high expressing genes in the
tumor associated with the GO term.
1. Spontaneous tumors were associated with
GO annotations less destructive in nature.
2. The genetically engineered tumors
exhibited significant gene functions related
to metastasis and immune detection,
hallmarks of an aggressive cancer behavior.
Sequence Data
(FASTQ)
FASTQC
TopHat
Cufflinks
Functional Annotation
Analysis with VLAD 3
Data quality assessment
using 10 different
measures
Alignment of sequencing
reads to the reference
genome for mouse
(GRCm38)
Association of mapped
reads to mouse gene
annotations from the
UCSC Genome Browser
Test for enrichment of
Biological Process terms
in the top 200 expressed
genes .
Sequence data from
normal and tumor
samples in FASTQ format
RNA Seq analysis was performed on 5 mouse lung tumors for this study. Two of the tumors
arose spontaneously and three were induced in genetically engineered mice using Cre
recombinase. All of the engineered mice carry a mutation in the KRAS gene, one of the
most commonly mutated genes in human lung cancer.
1) Goecks, J, Nekrutenko, A, Taylor, J and The Galaxy Team. Galaxy: a comprehensive approach
for supporting accessible, reproducible, and transparent computational research in the life
sciences. Genome Biol. 2010 Aug 25;11(8):R86
2) Mouse Genome Informatics; http://www.informatics.jax.org
3) VLAD http://proto.informatics.jax.org/prototypes/vlad-1.0.3/
GO: 0010941
regulation of
cell death
GO: 0043067
regulation of
programmed cell
death
GO: 0042981
regulation of
apoptotic process
GO:0019884
antigen
processing and
presentation of
exogenous antigen
GO:0002478
antigen
processing and
presentation of
exogenous
peptide antigen
VLAD output supports rapid discovery of biological processes
represented in tumor samples but not normal lung tissues and vice
versa. For example, ST31 (spontaneous bronchioloalveolar) genes
associated with antigen processing are highly expressed in tumor cells
but not in normal lung tissue from the same animal.