This study investigated the role of SCGB3A2 in oncogenic K-ras-induced lung cancer using Scgb3a2 knockout mice. Mice with single floxed Scgb3a2 (+/f) and double floxed Scgb3a2 (f/f) were bred with Ccsp-Cre;LSL-K-rasG12D mice to induce lung cancer. Results showed Scgb3a2 (f/f) mice had a shortened lifespan compared to Scgb3a2 (+/f) mice, suggesting SCGB3A2 inhibits K-ras-induced carcinogenesis. However, tumor number, size, and lung weight were similar between genotypes. This indicates SC

1. 21
BackgroundAbstract
Secretoglobin (SCGB) 3A2, formerly called
Uteroglobin-related protein (UGRP) 1, was
originally identified as a downstream target for a
homeodomain transcription factor, NKX2-1.
SCGB3A2 is highly expressed in the epithelial
cells of the trachea, bronchus and bronchioles.
SCGB3A2 exhibits anti-inflammatory and anti-
fibrotic functions as well as growth factor
activity that is responsible for fetal lung
development. Our recent study demonstrated
that SCGB3A2 is over-expressed in lung
carcinomas, particularly adenocarcinomas in
humans. In order to further understand the
physiological roles of SCGB3A2 in lungs and/or
lung diseases including lung cancer, we have
produced Scgb3a2 knockout mice. These
knockout mice are characterized to identify
additional roles for SCGB3A2 in lungs. Our
purpose of this study is to understand the
biological function and molecular mechanism of
SCGB3A2 in a mouse model of oncogenic K-ras-
induced lung cancer.
Ccsp-Cre/LSL-K-rasG12D knock-in mouse
mated with floxed Scgb3a2 mouse
Compare the phenotypes of Scgb3a2 (+/flox)
to Scgb3a2 (flox/flox) in oncogenic K-ras mice
Future Experiments
• To obtain pathological analysis for
the classification of cancer severity.
• To extend the observational time in
order to obtain a more accurate
survival curve and increase the
sample size of +/fl and fl/fl mice for
lung weight data.
• Prepare mouse embryonic (MEF) and
primary lung fibroblasts from mice
carrying K-rasG12D in the
background of Scgb3a2(-/-) or wild
type, and treat them with
recombinant adenovirus expressing
Cre recombinase (Ad-Cre) to study
the mechanism for the role of
SCGB3A2 in carcinogenesis.
Summary
• Floxed, transgenic and knock-in alleles
were identified by PCR.
• Oncogenic K-ras induced lung cancers
were developed in Scgb3a2 (+/flox),
Scgb3a2 (flox/flox) and wild type mice.
• Scgb3a2 (flox/flox) displays a
shortened life span compared with
Scgb3a2 (+/flox) in oncogenic K-ras-
induced lung carcinogenesis.
• Gross tumor number, body weight,
tumor size, and lung weight had no
significant difference between
Scgb3a2 (+/flox) and Scgb3a2
(flox/flox) mice.
• SCGB3A2 might inhibit oncogenic K-
ras-induced lung carcinogenesis.
Genotype PCR
650
400
200
WT +/f f/f
300
500
WT (411 bp)
Floxed (354 bp)
- - + +
550 bp
400
500
650
850
1000
408 bp
400
500
300
- - + +
Floxed Scgb3a2
Ccsp-Cre
K-ras G12D (Oncogenic K-ras)
LSL-K-rasG12D
knock-in allele
Floxed Scgb3a2X
WT K-ras allele
Activated K-rasG12D
allele
Ccsp-Cre;LSL-K-ras G12D
Effect of the loss of SCGB3A2 on oncogenic K-ras-induced lung
carcinogenesis in mice
Angela Zhou, Mitsuhiro Yoneda, Jorge Paiz, and Shioko Kimura
Endocrinology Section, Laboratory of Metabolism, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892, USA
SCGB3A2 suppresses K-ras-induced carcinogenesis in
mice lungs.
Ccsp-Cre;LSL-K-ras
G12D;Scgb3a2
single floxed (+/f)
Ccsp-Cre;LSL-K-ras
G12D;Scgb3a2
double floxed (f/f)
Expected incidence of
lung cancer
Hypothesis
Mice
Ccsp-Cre;LSL-K-rasG12D;Scgb3a2(f/f) and Ccsp-
Cre;LSL-K-rasG12D;Scgb3a2(+/f) mice are used in
this study. Ccsp-Cre/LSL-K-rasG12D conditional
mutant mice were kindly provided from Dr. DeMayo
(Baylor College of Medicine). Floxed-Scgb3a2 mice
were produced in our laboratory. All mice were
maintained under standard specific-pathogen-free
conditions. All experiments were carried out
following guidelines for animal use issued by the
National Institutes of Health and approved by the
National Cancer Institute (NCI) Animal Care and Use
Committee.
Genotyping for conditional mutant mice
Floxed, transgenic and knock-in mice were identified
using PCR primers specific for each transgene.
Amplification of PCR products for floxed and Cre
transgenic mice were performed as follows:
•Denaturation at 94°C for 5 min
•38 cycles of:
•Denaturation at 94°C for 15 sec
•Annealing at 60°C for 30 sec
•Extension at 72°C for 15 sec
•5-min extension at 72°C
Amplification for the LSL-K-rasG12D knock-in allele
was performed as follows:
•Denaturation at 94°C for 3 min
•40 cycles of:
•Denaturation at 94°C for 30 sec
•Annealing at 58°C for 90 sec
•Extension at 72°C for 60 sec
•5-min extension at 72°C
H&E Staining
Lung tissues were fixed in 10% buffered formalin,
embedded in paraffin and cut into 5-µm sections.
Sections were deparaffinized and stained with
hematoxylin and eosin (H&E).
Methods
Ccsp-Cre;LSL-K-ras G12D;Scgb3a2 single floxed (+/f)
Lung Tumors in 6-month-old
male oncogenic K-ras mice
*Blue arrows indicate lung tumors
Ccsp-Cre;LSL-K-ras G12D;Scgb3a2 double floxed (f/f)
Acknowledgments
• Yan Cai
• Manabu Iwadate
• Shuko Kawabe
• Tsubasa Murata
• Lei Xu
X40
Ccsp-Cre;LSL-K-ras G12D;
Scgb3a2 single floxed (+/f)
Ccsp-Cre;LSL-K-ras G12D;
Scgb3a2 double floxed (f/f)
C; Cancerous tissue
Normal
X400 X400
X40 X40
X400
c
c
c
Survival Curve - Male
Results
p value = 0.035
Immunohistochemistry
Ccsp-Cre;LSL-K-ras G12D;
Scgb3a2 single floxed (+/f)
Ccsp-Cre;LSL-K-ras G12D;
Scgb3a2 double floxed (f/f)
*Brown indicates the expression of SCGB3A2.
Ccsp-Cre;LSL-K-ras G12D;
Wild Type
c
•SCGB3A2 was not
expressed in Ccsp-
Cre;LSL-K-ras G12D;
Scgb3a2 double floxed
(f/f).
c c
• Shigetoshi Yokoyama
• NIH Summer Intern
Program
H&E Staining of adenocarcinomas in
oncogenic K-ras lungs
C; Cancerous tissue