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. 2017 Oct 15;141(8):1589-1599.
doi: 10.1002/ijc.30851. Epub 2017 Jul 17.

Development of Kras mutant lung adenocarcinoma in mice with knockout of the airway lineage-specific gene Gprc5a

Junya Fujimoto  1 , Sayuri Nunomura-Nakamura  1   2 , Yihua Liu  3 , Wenhua Lang  1 , Tina McDowell  1 , Yasminka Jakubek  3 , Dalia Ezzeddine  4 , Joshua Kapere Ochieng  1 , Jason Petersen  5 , Gareth Davies  5 , Junya Fukuoka  2 , Ignacio I Wistuba  1 , Erik Ehli  5 , Jerry Fowler  3 , Paul Scheet  3 , Humam Kadara  1   6
Affiliations

Development of Kras mutant lung adenocarcinoma in mice with knockout of the airway lineage-specific gene Gprc5a

Junya Fujimoto et al. Int J Cancer. .

Abstract

Despite the urgency for prevention and treatment of lung adenocarcinoma (LUAD), we still do not know drivers in pathogenesis of the disease. Earlier work revealed that mice with knockout of the G-protein coupled receptor Gprc5a develop late onset lung tumors including LUADs. Here, we sought to further probe the impact of Gprc5a expression on LUAD pathogenesis. We first surveyed GPRC5A expression in human tissues and found that GPRC5A was markedly elevated in human normal lung relative to other normal tissues and was consistently downregulated in LUADs. In sharp contrast to wild-type littermates, Gprc5a-/- mice treated chronically with the nicotine-specific carcinogen NNK developed LUADs by 6 months following NNK exposure. Immunofluorescence analysis revealed that the LUADs exhibited abundant expression of surfactant protein C and lacked the clara cell marker Ccsp, suggesting that these LUADs originated from alveolar type II cells. Next, we sought to survey genome-wide alterations in the pathogenesis of Gprc5a-/- LUADs. Using whole exome sequencing, we found that carcinogen-induced LUADs exhibited markedly higher somatic mutation burdens relative to spontaneous tumors. All LUADs were found to harbor somatic mutations in the Kras oncogene (p. G12D or p. Q61R). In contrast to spontaneous lesions, carcinogen-induced Gprc5a-/- LUADs exhibited mutations (variants and copy number gains) in additional drivers (Atm, Kmt2d, Nf1, Trp53, Met, Ezh2). Our study underscores genomic alterations that represent early events in the development of Kras mutant LUAD following Gprc5a loss and tobacco carcinogen exposure and that may constitute targets for prevention and early treatment of this disease.

Keywords: Gprc5a; Kras; carcinogenesis; lung adenocarcinoma; whole-exome sequencing.

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Conflict of interest statement

The authors report no conflicts of interests.

Figures

Figure 1
Figure 1. GPRC5A expression patterns in human normal and malignant lung tissues
A. Expression levels of GPRC5A mRNA were assessed in an array set comprised of human pan-normal samples (GSE7307 from the gene expression omnibus) as described in Supplementary Methods and in datasets consisting of human LUADs. Normal tissues were ranked (from top to bottom) by decreasing log (base 2) median-centered expression of GPRC5A. B. GPRC5A mRNA expression was also surveyed in datasets - including a set we previously reported consisting of human LUADs and uninvolved normal tissues. Boxes represent 25% - 75% expression ranges and whiskers constitute maxima and minima. Solid horizontal lines represent median GPRC5A mRNA log (base 2) expression values. P-values were obtained using t-tests.
Figure 2
Figure 2. Tobacco carcinogen-mediated LUAD pathogenesis in the Gprc5a-/- mouse
A. Schematic depicting timeline of NNK (three times 50mg/kg body weight per week for eight weeks) or saline treatment of eight weeks old Gprc5a-/- mice (see Methods). Mice were divided into groups of five to six mice (per treatment and time point) and were sacrificed at every month for seven months following saline and NNK treatment. B. At each of the indicated time points, mice lungs were histopathologically evaluated for the development of lesions. The lesions were pathologically categorized (hyperplasias, adenomas and adenocarcinomas) based on previously reported guidelines for murine lung lesions (23) and were then enumerated and compared and contrasted across time points and between treatment groups. C. LUAD burdens, indicated by the number of LUADs per mouse, were statistically compared and contrasted at six and seven months following NNK treatment.
Figure 3
Figure 3. Immunofluorescence analysis of airway lineage markers in lung lesions and normal tissues
Immunofluorescence (IF) analysis of airway lineage markers of Clara (Ccsp) and alveolar type 2 (AT2) (Sftpc) cells was performed in Gprc5a-/- lesions and adjacent normal regions as described in Materials and Methods. B. IF analysis of Gprc5a and the AT1 cell marker Pdpn was performed as described in Materials and Methods. All images were captured using a BX61 immunofluorescent system (Olympus) and merged using the CytoVision workstation (Leica biosystems Inc.) at a magnification of 200× (scale bar denoting 100 μm).
Figure 4
Figure 4. Whole-exome sequencing analysis of spontaneous and NNK-derived Gprc5a-/- LUADs
Two spontaneous (> 16 months following saline) LUADs from two Gprc5a-/- mice as well as five LUADs that developed in different mice by five to seven months following NNK treatment were analyzed by whole-exome sequencing (WES) as described in Materials and Methods. Somatic calls were contrasted against whole-exomes from three tail veins (from two Gprc5a-/- and one WT mice) and Gprc5a-/- normal lung tissues at one month following saline (see Methods). Single nucleotide variants (SNVs) were prioritized based on variants occurring in bona fide driver genes . The top panel depicts total number of silent (syn) and non-silent (nonsyn) exonic mutations per LUAD. In the lower panel, columns represent LUADs and rows represent mutated driver genes. Arrow indicates somatic Kras (p.G12D or p.Q61R) mutations.
Figure 5
Figure 5. Genome-wide copy number variation in spontaneous and NNK-derived Gprc5a-/- LUADs
Copy number variations (CNVs) were assessed from sequencing data and read depth using FREEC and Sequenza as described in the Supplementary Methods. Read depth was analyzed in LUADs relative to depth in normal samples to estimate copy number in 8 kb windows followed by segmentation via a LASSO-based algorithm. Genome-wide CNAs across all LUADs were then plotted. Loss, blue; gain, red; not discernable, black.
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