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Transcriptome Analysis of Spontaneous and Induced Lung Tumors in Mice using RNA Seq2Janaya 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

ST45Normal 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.