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Smoking perpetuates in cytologically normal airways a molecular “field of injury” that is pertinent to lung cancer and early detection. The evolution of airway field changes prior to lung oncogenesis is poorly understood largely due to the long latency of lung cancer in smokers. Here, we studied airway expression changes prior to lung cancer onset in mice with knockout of the Gprc5a gene (Gprc5a−/−) and tobacco carcinogen (NNK) exposure and that develop the most common type of lung cancer, lung adenocarcinoma, within 6 months following exposure. Airway epithelial brushings were collected from Gprc5a−/− mice before exposure and at multiple times post-NNK until time of lung adenocarcinoma development and then analyzed by RNA sequencing. Temporal airway profiles were identified by linear models and analyzed by comparative genomics in normal airways of human smokers with and without lung cancer. We identified significantly altered profiles (n = 926) in the NNK-exposed mouse normal airways relative to baseline epithelia, a subset of which were concordantly modulated with smoking status in the human airway. Among airway profiles that were significantly modulated following NNK, we found that expression changes (n = 22) occurring as early as 2 months following exposure were significantly associated with lung cancer status when examined in airways of human smokers. Furthermore, a subset of a recently reported human bronchial gene classifier (Percepta; n = 56) was enriched in the temporal mouse airway profiles. We underscore evolutionarily conserved profiles in the normal-appearing airway that develop prior to lung oncogenesis and that comprise viable markers for early lung cancer detection in suspect smokers. Cancer Prev Res; 11(4); 237–48. ©2018 AACR.

Supplementary Tables Table S1. Early efffcts of NNK exposure on the mouse airway transcriptome. Table S2. Evolutionarily conserved decreased gene expression in the airway following NNK exposure. Table S3. Genes differentially regulated in the mouse airway at the indicated timepoints post NNK exposure. Table S4. Mouse genes downregulated as early as T2 vs T0 and enriched in airways of human smokers with lung cancer relative to cancer-free smokers. Table S5. Pathways analysis of Mouse genes enriched in airways of human smokers with lung cancer and modulated during in vivo lung oncogenesis

Supplementary Methods and Supplementary Figure Legends

S1. Lung tumor development in NNK-exposed Gprc5a-/- mice. S2. Evolutionarily conserved patterns of tobacco-carcinogen mediated gene expression in murine and human airway epithelia. S3. Early and persistent changes in mouse airway gene expression in vivo following tobacco carcinogen exposure and during lung oncogenesis. S4. Gene profiles downregulated in the mouse normal-appearing airway field of injury are overall suppressed in lung tumors relative to adjacent normal lung.

Rationale: Early pathogenesis of lung adenocarcinoma (LUAD) remains largely unknown. We found that, relative to wild-type littermates, the innate immunomodulator Lcn2 (lipocalin-2) was increased in...

Smoking perpetuates in cytologically normal airways a molecular “field of injury” that is pertinent to lung cancer and early detection. The evolution of airway field changes prior to lung oncogenesis is poorly understood largely due to the long latency of lung cancer in smokers. Here, we studied airway expression changes prior to lung cancer onset in mice with knockout of the Gprc5a gene (Gprc5a−/−) and tobacco carcinogen (NNK) exposure and that develop the most common type of lung cancer, lung adenocarcinoma, within 6 months following exposure. Airway epithelial brushings were collected from Gprc5a−/− mice before exposure and at multiple times post-NNK until time of lung adenocarcinoma development and then analyzed by RNA sequencing. Temporal airway profiles were identified by linear models and analyzed by comparative genomics in normal airways of human smokers with and without lung cancer. We identified significantly altered profiles (n = 926) in the NNK-exposed mouse normal airways relative to baseline epithelia, a subset of which were concordantly modulated with smoking status in the human airway. Among airway profiles that were significantly modulated following NNK, we found that expression changes (n = 22) occurring as early as 2 months following exposure were significantly associated with lung cancer status when examined in airways of human smokers. Furthermore, a subset of a recently reported human bronchial gene classifier (Percepta; n = 56) was enriched in the temporal mouse airway profiles. We underscore evolutionarily conserved profiles in the normal-appearing airway that develop prior to lung oncogenesis and that comprise viable markers for early lung cancer detection in suspect smokers. Cancer Prev Res; 11(4); 237–48. ©2018 AACR.

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.

Relative to other lung adenocarcinomas (LUADs), Kras mutant LUAD displays dismal prognosis warranting the need for new strategies for early treatment of this lung cancer subtype. Limiting these advances is our poor understanding of events that drive Kras mutant LUAD development. In efforts to fill this void, we recently found that mice with knockout of G-protein coupled receptor 5A (Gprc5a-/-) and after tobacco carcinogen exposure (nicotine-specific nitrosamine ketone/NNK) developed LUADs harboring driver somatic Kras mutations. To understand early events preceding the onset of these Kras mutant LUADs, we performed RNA-sequencing (RNA-Seq) of normal airways in Gprc5a-/- mice and wild type (WT) littermates prior to NNK exposure. We found markedly elevated expression of the antimicrobial lipocalin 2 (Lcn2) gene in normal airways of Gprc5a-/- mice. Also, analysis of publicly available human datasets revealed elevated expression of LCN2 in human LUADs relative to normal lung tissues particularly in KRAS mutant tumors. We then sought to probe the role of Lcn2 in the pathogenesis of Kras mutant LUAD. Mice with knockout of host Lcn2 (Gprc5a-/-/Lcn2-/-) showed increased lung tumor development compared to Gprc5a-/- littermates. RNA-Seq of whole lungs at baseline and at seven months post-NNK (n = 9-10 mice each group) pinpointed differential expression profiles that denoted decreased anti-tumor immunity in Gprc5a-/-/Lcn2-/- mice. Computational estimation of immune infiltrates demonstrated increased levels of neutrophils post-NNK in Gprc5a-/-/Lcn2-/- mice but not in Gprc5a-/-littermates. By flow cytometry analysis of lung tissues, we found elevated numbers of various lymphoid and myeloid immune cells in both groups following NNK exposure and increased neutrophil counts in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- littermates. Histopathological assessment also revealed increased numbers of pro-inflammatory lesions in mice lacking Lcn2. Gprc5a-/-/Lcn2-/- mice at three months post-NNK were also found to exhibit increased production of IL-17A by lung-resident CD4+ and CD8+ T cells relative to Gprc5a-/- littermates concomitant with decreased expression of the anti-tumor Th1/cytolytic immune markers Ifnγ and Gzmb. Preliminary analysis also showed elevated expression of the major immune checkpoint PD-L1 on alveolar macrophages in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- mice with WT Lcn2. Lastly, we preliminarily found, by bacterial 16S rRNA sequencing of temporally collected fecal samples, globally altered microbiome profiles in mice lacking Lcn2. Our findings underscore novel cues in lung oncogenesis and suggest that Lcn2 decreases anti-tumor immunity, perturbs the host microbiome and promotes development of Kras mutant LUAD thereby offering new venues for early treatment of this fatal malignancy. Citation Format: Maya Hassane, Warapen Treekitkarnmongkol, Tina L. McDowell, Smruthy Sivakumar, Wenhua Lang, Joshua K. Ochieng, Sayuri Nunomura-Nakamura, Casey Finnicum, Christel Davis, Gareth E. Davies, Junya Fukuoka, Tina Cascone, Florencia McAllister, Ignacio I. Wistuba, Erik Ehli, Seyed J. Moghaddam, Paul Scheet, Junya Fujimoto, Humam Kadara. Host lipocalin 2 protects against Kras mutant lung cancer development by maintaining an anti-tumor immune contexture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4952.