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Supplementary file containing Supplementary Methods (pertaining to reagents, imaging, immunoblotting, cellular studies, microarray data, NF-κB ELISA, PCR, cell culture, histology, cytology, flow cytometry, constructs, and cellular Assays), 4 Supplementary Tables (pertaining to number of experimental mice, PCR primers, antibodies, and lentiviral shRNA pools), and 7 Supplementary Figures (described in the main and Supplementary text).

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB–activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D. Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of RelB, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS-mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo. IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS-mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease.Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939–51. ©2018 AACR.

Introduction: cAMP response element-binding protein (CREB) mediates proliferative and inflammatory transcription in neurodegeneration and cancer, but its role in malignant immune-evasion is obscure. Objectives: To discover the mechanisms underlying the oncogenic properties of CREB in KRAS-mutant lung adenocarcinoma (LUAD). Materials and Methods: We used conditional deletion, focal overexpression, and pharmacologic inhibition of CREB in different mouse and cellular models of early-stage and metastatic KRAS-mutant LUAD. We determined the neutrophil (ΝΦ) influx in the bronchoalveolar lavage (BAL) from LUAD-affected conditionally Creb1-deleted lungs. We employed The Cancer Genome Atlas, the GEO dataset GSE43458, and the human protein atlas to examine CREB and CXCR1 in human LUAD. Results: CREB is overexpressed in murine KRAS-mutant LUAD, pulmonary deletion of Creb1 (encoding murine CREB) inhibits LUAD development, and Creb1-overexpression augments the tumorigenicity of KRAS-mutant cells. Conditional Creb1 deletion in KRAS -mutant LUAD cells causes overexpression of CXCR1 ligands, and LUAD-bearing Creb1-deleted mice display increased pulmonary ΝΦ. Cxcr1-deficient mice are selectively permissive to KRAS-mutant tumor growth and show defective ΝΦ recruitment. The pro-tumor effects of CREB require intact host Cxcr1 and those of host Cxcr1 necessitate mutant KRAS in cancer cells. Pharmacologic CREB blockade prevents tumor growth and restores ΝΦ recruitment only when initiated before immune-evasion of KRAS-mutant LUAD. CREB and CXCR1 expression of human LUAD are compartmentalized to tumor and stromal cells, respectively, while CREB-controlled genes profoundly impact survival. Conclusions: CREB-mediated immune evasion of KRAS-mutant LUAD rests on signaling to ΝΦ CXCR1 and is actionable.

cAMP response element-binding protein (CREB) mediates proliferative and inflammatory gene transcription in neurodegeneration and cancer, but its role in malignant immune-evasion of lung adenocarcinoma (LUAD) is unknown. We show that human LUAD of smokers are frequently altered along the CREB pathway and we employ mouse models to discover that KRAS-mutant LUAD co- opt CREB to evade immune rejection by tumoricidal neutrophils. For this, KRAS- driven CREB activation suppresses CXC-chemokine expression and prevents recruitment of CXCR1+ neutrophils. CREB1 is shown to be pro-tumorigenic in five different LUAD models, a function that is dependent on host CXCR1. Pharmacologic CREB blockade prevents tumor growth and restores neutrophil recruitment only when initiated before immune-evasion of KRAS-mutant LUAD. CREB and CXCR1 expression in human LUAD are compartmentalized to tumor and stromal cells, respectively, while CREB-regulated genes and neutrophils impact survival. In summary, CREB-mediated immune evasion of KRAS-mutant LUAD relies on signaling to neutrophil CXCR1 and is actionable.

BackgroundSurvival after curative resection of early-stage lung adenocarcinoma (LUAD) varies and prognostic biomarkers are urgently needed.MethodsLarge-format tissue samples from a prospective cohort of 200 patients with resected LUAD were immunophenotyped for cancer hallmarks TP53, NF1, CD45, PD-1, PCNA, TUNEL and FVIII, and were followed for a median of 2.34 (95% CI 1.71–3.49) years.ResultsUnsupervised hierarchical clustering revealed two patient subgroups with similar clinicopathological features and genotype, but with markedly different survival: “proliferative” patients (60%) with elevated TP53, NF1, CD45 and PCNA expression had 50% 5-year overall survival, while “apoptotic” patients (40%) with high TUNEL had 70% 5-year survival (hazard ratio 2.23, 95% CI 1.33–3.80; p=0.0069). Cox regression and machine learning algorithms including random forests built clinically useful models: a score to predict overall survival and a formula and nomogram to predict tumour phenotype. The distinct LUAD phenotypes were validated in The Cancer Genome Atlas and KMplotter data, and showed prognostic power supplementary to International Association for the Study of Lung Cancer tumour–node–metastasis stage and World Health Organization histologic classification.ConclusionsTwo molecular subtypes of LUAD exist and their identification provides important prognostic information.

The mechanisms tumor cells use to hijack the immune system are largely uncharted. Here we used bioluminescent nuclear factor (NF)-κB reporter mice and macrophages to discover that metastatic tumors trigger NF-κB activation in host macrophages, dependent on mutant KRAS signaling and delivered via secretory versican. Versican activates NF-κB in tumor-associated macrophages via inhibitor of NF-κB kinase (IKK) β, resulting in release of interleukin (IL)-1β into the tumor microenvironment. Versican silencing in cancer cells or conditional IKKβ deletion in macrophages prevents myeloid NF-κB activation and metastasis. Versican is overexpressed and/or mutated in human cancers and metastatic effusions with KRAS mutations, predicts poor survival, can aid in the development of diagnostic platforms for pleural metastasis, and is druggable via toll-like receptor (TLR) 1/2 inhibition. The data indicate a cardinal role for tumor-derived versican in establishing cross-talk with macrophage IKKβ during metastasis and may foster the development of new therapies and diagnostic tools.

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide. Nevertheless, syngeneic mouse models of the disease are sparse, and cell lines suitable for transplantable and immunocompetent mouse models of LADC remain unmet needs. We established multiple mouse LADC cell lines by repeatedly exposing two mouse strains (FVB, Balb/c) to the tobacco carcinogens urethane or diethylnitrosamine and by culturing out the resulting lung tumours for prolonged periods of time. Characterization of the resulting cell lines (n = 7) showed that they were immortal and phenotypically stable in vitro, and oncogenic, metastatic and lethal in vivo. The primary tumours that gave rise to the cell lines, as well as secondary tumours generated by transplantation of the cell lines, displayed typical LADC features, such as glandular architecture and mucin and thyroid transcription factor 1 expression. Moreover, these cells exhibited marked molecular similarity with human smokers’ LADC, including carcinogen-specific Kras point mutations (KrasQ61R in urethane- and KrasQ61H in diethylnitrosamine-triggered cell lines) and Trp53 deletions and displayed stemness features. Interestingly, all cell lines overexpressed proliferin, a murine prolactin orthologue, which functioned as a lung tumour promoter. Furthermore, prolactin was overexpressed and portended poor prognosis in human LADC. In conclusion, we report the first LADC cell lines derived from mice exposed to tobacco carcinogens. These cells closely resemble human LADC and provide a valuable tool for the functional investigation of the pathobiology of the disease.

International audience; A comprehensive characterization of lung adenocarcinoma (LADC) clinical features is currently missing. We prospectively evaluated Caucasian patients with early-stage LADC. Patients with LADC diagnosed between 2011 and 2015 were prospectively assessed for lung resection with curative intent. Fifty clinical, pathologic, radiologic, and molecular variables were recorded. Patients were followed till death/study conclusion. The main findings were compared to a separate cohort from France. Of 1943 patients evaluated, 366 were enrolled (18.8%; 181 female; 75 never-smokers; 28% of registered Bavarian cases over the study period). Smoking and obstruction were significantly more prevalent in GLAD compared with adult Bavarians (P < 0.0001). Ever-smoker tumors were preferentially localized to the upper lobes. We observed 120 relapses and 74 deaths over 704 cumulative follow-up years. Median overall and disease-free survival were >7.5 and 3.6 years, respectively. Patients aged 65 years, resected >60 days postdiagnosis, with abnormal FVC/DLCO VA , N2/N3 stage, or solid histology had significantly decreased survival estimates. These were fit into a weighted locoregional LADC death risk score that outperformed pTNM7 in predicting survival in the GLAD and in our second cohort. We define the clinical gestalt of locoregional LADC and provide a new clinical tool to predict survival, findings that may aid future management and research design

Introduction: cAMP response element-binding protein (CREB) mediates proliferative and inflammatory transcription in neurodegeneration and cancer, but its role in malignant immune-evasion is obscure. Objectives: To discover the mechanisms underlying the oncogenic properties of CREB in KRAS-mutant lung adenocarcinoma (LUAD). Materials and Methods: We used conditional deletion, focal overexpression, and pharmacologic inhibition of CREB in different mouse and cellular models of early-stage and metastatic KRAS-mutant LUAD. We determined the neutrophil (ΝΦ) influx in the bronchoalveolar lavage (BAL) from LUAD-affected conditionally Creb1-deleted lungs. We employed The Cancer Genome Atlas, the GEO dataset GSE43458, and the human protein atlas to examine CREB and CXCR1 in human LUAD. Results: CREB is overexpressed in murine KRAS-mutant LUAD, pulmonary deletion of Creb1 (encoding murine CREB) inhibits LUAD development, and Creb1-overexpression augments the tumorigenicity of KRAS-mutant cells. Conditional Creb1 deletion in KRAS -mutant LUAD cells causes overexpression of CXCR1 ligands, and LUAD-bearing Creb1-deleted mice display increased pulmonary ΝΦ. Cxcr1-deficient mice are selectively permissive to KRAS-mutant tumor growth and show defective ΝΦ recruitment. The pro-tumor effects of CREB require intact host Cxcr1 and those of host Cxcr1 necessitate mutant KRAS in cancer cells. Pharmacologic CREB blockade prevents tumor growth and restores ΝΦ recruitment only when initiated before immune-evasion of KRAS-mutant LUAD. CREB and CXCR1 expression of human LUAD are compartmentalized to tumor and stromal cells, respectively, while CREB-controlled genes profoundly impact survival. Conclusions: CREB-mediated immune evasion of KRAS-mutant LUAD rests on signaling to ΝΦ CXCR1 and is actionable.

Background: Respiratory epithelial dynamics and molecular signatures participating in adult lung maintenance and chemical carcinogenesis need to be clearly identified. Aims: We used mouse models of lung epithelial fate to chart the contribution of the respective cell lineages in the mouse lung during aging and after challenge with noxious and carcinogenic insults. Methods: We generated lung cell fate reporter mice by crossing Scgb1a1.Cre, Sftpc.Cre and Lyz2.Cre mice with the mT/mG Cre-reporter mice, resulting in expression of GFP in all Cre-recombined cells. These reporter mice were then subjected to various noxious (bleomycin and naphthalene administration at 0.08 units and 250 mg/kg, respectively; n≥10 mice/group) and carcinogenic insults (urethane administration at 1 g/Kg; n≥10 mice/group). The GFP-labelled cells were then monitored in the injured lungs and the developed adenocarcinomas. Results: We observed that lung adenocarcinomas were spatially linked to neighboring airways. The cells of all tumors of GFP;Sftpc.Cre mice and all cells of ~50% of GFP;Lyz2.Cre tumors were GFP+. Interestingly, a significant cell proportion of all tumors of GFP;Scgb1a1.Cre mice were GFP+, indicating a bronchial origin. Furthermore, the airway-specific signature was redistributed to the alveoli after toxic insults and resulted in marked contributions of airway-labeled cells to injury-recovered alveoli. Our results also indicate that airway cells maintain Kras mutations, possibly contributing to lung cancer initiation. Conclusions: Our findings identify an involvement of airway molecular programs in alveolar maintenance and carcinogen-induced lung adenocarcinomas. Funding General Secretariat for Research and Technology (GSRT) and Hellenic Foundation for Research and Innovation (HFRI) grant #1853.

Lung cancer is the most lethal cancer worldwide, and its epidemic appears to be on a continuous rise despite smoking prevention and cessation programs. It is generally believed that inflammation alters the microenvironment of tobacco carcinogen-mutated respiratory epithelial cells, fostering their survival and sustained growth instead of their eradication. Macrophages are inflammatory leukocytes that play key and contradicting roles in tumor initiation and progression, but the reason for their divergent functions in cancer remain elusive. This study is designed in order to clarify macrophages’ role during the development of lung adenocarcinoma. To investigate this, we employed mouse models of early adenocarcinoma induction, in parallel with in vivo models of adenocarcinoma growth and metastasis. The aforementioned models have been combined with genetically modified animal models of macrophage localization and ablation to study the recruitment of these cells to tumors as well as their interaction with cancer cells at different stages of the disease. Moreover, we developed in vitro and utilized in vivo different macrophage types to clarify their distinctive roles in lung adenocarcinoma progression. Our results indicate that macrophages are actively recruited to lung tumors and may play contradicting roles in lung adenocarcinoma development. Although, GM-CSF macrophages function as innocent bystanders, M-CSF macrophages induce both tumor initiation in the respiratory epithelium and the progression of the disease. M-CSF macrophages were found here to fuel KRAS-mutant lung adenocarcinoma formation and growth in mice by secreting IL-1β setting a rational framework for further study of macrophage functions in lung tumors.

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide and is largely inflicted by carcinogens contained in tobacco smoke. The generation of cell lines mimicking traits of human LADC will profoundly advance our understanding of the pathobiology of the disease, as they offer an easy and valuable tool to study the cellular and molecular aspects of carcinogenesis. Here we describe a detailed protocol for the generation of such cell lines, following the exposure of experimental mouse strains to different tobacco carcinogens and isolation of the resulting lung tumors.

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide and is largely inflicted by carcinogens contained in tobacco smoke. The generation of cell lines mimicking traits of human LADC will profoundly advance our understanding of the pathobiology of the disease, as they offer an easy and valuable tool to study the cellular and molecular aspects of carcinogenesis. Here we describe a detailed protocol for the generation of such cell lines, following the exposure of experimental mouse strains to different tobacco carcinogens and isolation of the resulting lung tumors.

Malignant pleural mesothelioma (MPM) arises from mesothelial cells lining the pleural cavity of asbestos-exposed individuals and rapidly leads to death. MPM harbors loss-of-function mutations in BAP1, NF2, CDKN2A, and TP53, but isolated deletion of these genes alone in mice does not cause MPM and mouse models of the disease are sparse. Here, we show that a proportion of human MPM harbor point mutations, copy number alterations, and overexpression of KRAS with or without TP53 changes. These are likely pathogenic, since ectopic expression of mutant KRAS(G12D) in the pleural mesothelium of conditional mice causes epithelioid MPM and cooperates with TP53 deletion to drive a more aggressive disease form with biphasic features and pleural effusions. Murine MPM cell lines derived from these tumors carry the initiating KRAS(G12D) lesions, secondary Bap1 alterations, and human MPM-like gene expression profiles. Moreover, they are transplantable and actionable by KRAS inhibition. Our results indicate that KRAS alterations alone or in accomplice with TP53 alterations likely play an important and underestimated role in a proportion of patients with MPM, which warrants further exploration., European Union (EU); Horizon 2020; European Research Council 2010 Starting Independent Investigator; ERC Proof of Concept 2015 (ERC-PoC-2015); German Research Society; ALTERNATIVE; German Ministry for Education and Research; German Center for Lung Research Translational Research Grant; Greek State Scholarship Foundation Program; European Union (EU); European Union Social Fund; Greek National Fund; Reinforcement of Postdoctoral Researchers-1 stand 2nd cycles; General Secretariat for Research and Innovation; Hellenic Foundation for Research and Innovation; REPSIRE European Respiratory Society Fellowship; Institut National de la Sante et de la Recherche Medicale (INSERM); Centre National de la Recherche Scientifique (CNRS); Institut de Recherche en Sante Respiratoire des Pays de la Loire; National Research Agency Programme d' Investissements d' Avenir; Pays de la Loire Region Research Program; Ligue Contrele Cancer; Chancellerie des Universites de Paris; Projekt DEAL

Malignant pleural mesothelioma (MPM) arises from mesothelial cells lining the pleural cavity of asbestos-exposed individuals and rapidly leads to the development of pleural effusion and death. MPM harbours loss-of-function mutations in genes likeBAP1, NF2, CDKN2A, andTP53, but isolated deletion of these genes alone in mice does not cause MPM and mouse models of the disease are sparse. Here we show that a significant proportion of human MPM harbour point mutations and copy number alterations in theKRASproto-oncogene. These mutations are likely pathogenic, since ectopic expression of mutantKRASG12Din the pleural mesothelium of conditional mice causes MPM. Murine MPM cell lines derived from these tumours carry the initiatingKRASG12Dlesions, secondaryBap1alterations, and human MPM-like gene expression profiles. Moreover, they are transplantable and actionable by KRAS inhibition. Our results indicate thatKRASmutations likely play an important and underestimated role in MPM, which warrants further exploration.

ImportancePleural effusions frequently signal disseminated cancer. Diagnostic markers of pleural malignancy at presentation that would assess cancer risk and would streamline diagnostic decisions remain unidentified.ObjectiveThe present study aimed at identifying and validating predictors of malignant pleural effusion at patient presentation.DESIGN, SETTING, AND PARTICIPANTSA consecutive cohort of 323 patients with pleural effusion (PE) from different etiologies were recruited between 2013-2017 and was retrospectively analyzed. Data included history, chest X-ray, and blood/pleural fluid cell counts and biochemistry. Group comparison, receiver-operator characteristics, unsupervised hierarchical clustering, binary logistic regression, and random forests were used to develop the malignant pleural effusion detection (MAPED) score. MAPED was validated in an independent retrospective UK cohort (n= 238).Main Outcomes and MeasuresThe outcome was diagnostic of pleural effusion in patients, and the clinical and laboratory indicators available of the patient were measured.ResultsFive variables showed significant diagnostic power and were incorporated into the 5-point MAPED score. Age > 55 years, effusion size > 50% of the most affected lung field, pleural neutrophil count < 2,500/mm3, effusion protein > 3.5 g/dL, and effusion lactate dehydrogenase > 250 U/L, each scoring one point, predicted underlying cancer with the area under curve(AUC) = 0.819 (sensitivity=82%, specificity=74%,P< 10-15) in the derivation cohort. The AUC and net reclassification improvement (NRI) of MAPED score and cytology were not significantly different. However, the integrated discrimination improvement (IDI) of The MAPED score displayed a slight increment(PConclusionsThe MAPED score identifies malignant pleural effusions with satisfactory accuracy and can be used complementary to cytology to streamline diagnostic procedures.

The Gauting lung adenocarcinoma donors cohort already established that the age and lung functions influence patients´ survival (1). Here, we conducted a retrospective, exploratory combination of clinical information with tumor tissue profiles in 200 patients of this cohort. Relevant tumor mutations (KRAS, EGFR, p53, and NF1), tissue profiling (angiogenesis, inflammation, apoptosis and proliferation rates) were quantified for all patients. Then, 100 patients were randomly selected for a training sub-cohort. Mutation status, tissue profiling and clinical variables (age, sex, body mass index, lung functions, TNM descriptors, tumor location, and histology) were analyzed for correlation with the smoking status. The tumors from smokers positively correlate with KRAS mutation, high body mass index, and proliferation. They also negatively correlate with EGFR and p53 mutations, high lung functions, and angiogenesis. In the training sub-cohort, these variables can discriminate the smoking status of 88% tumors. Those variables were used to analyze a second sub-cohort of 100 other patients and were able to identify the smoking status in 86 percent of the cases. In conclusion, we identified an association of molecular biomarkers and clinical variables that highlight novel tumor genome‐phenome links. This association also discriminates the smoking status of tumors indicating two distinct pathways of carcinogenesis evolution in the lungs. (1) Klotz LV, et al. Comprehensive clinical profiling of the Gauting locoregional lung adenocarcinoma donors. Cancer Med. 2019 Apr;8(4):1486-1499.

Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive KRAS mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease., eLife digest The deadliest form of lung cancer is called lung adenocarcinoma, or LUAD. Tobacco chemicals often cause the disease by damaging the genetic information of lung cells. The damage leads to harmful changes in the DNA sequence which prompt the cells to form tumors. For instance, the most common of these changes takes place in a gene called KRAS. However, it is still unclear exactly which type of lung cells are more likely to develop into a tumor. In the lungs, airway epithelial cells cover the inside of the passages that bring the air inside little sacks called alveoli, which are lined by alveolar cells. Previous studies have used genetic methods to switch on the KRAS mutation in different compartments of the mouse lung. This showed that groups of airway cells, of alveolar cells, and of a class of cells located at the junction between airways and alveoli could all give rise to cancer. However, these experiments did not examine how tobacco chemicals could give rise to tumors in different groups of lung cells. Here, Spella et al. triggered LUAD in adult mice by exposing them to the toxic chemicals found in tobacco smoke, but without making any change to the KRAS gene. These mice also had genetically engineered reporters that could be used to deduce where the resulting tumors came from. DNA sequencing showed that the airway epithelial cells gained KRAS mutations after the chemical treatment. When the airway epithelial cells were experimentally removed before the treatments with tobacco chemicals, these mice did not get LUAD tumors. Spella et al. also observed that the tobacco-induced tumors came from the epithelial cells in the airways, and not from the cells in the alveoli. Moreover, when the lung was damaged, airway cells could move to the alveoli and start adopting the identity of alveolar cells, thereby replenishing this population. Together, these experiments imply that tobacco-induced LUAD starts in the airway epithelial cells. These findings suggest that airway epithelial cells could be targeted to stop lung cancer early on. Further studies should also examine how airway epithelial cells can transition to look more like alveolar cells when the lungs get harmed.

Osteopontin (secreted phosphoprotein 1; SPP1) expression has been identified in human lung cancer and has been linked with enhanced tumour progression. To examine its functional role, we induced lung tumours by repetitive urethane or MCA/BHT lung carcinogens in C57BL/6 mice lacking both ( Spp1-/- ), one ( Spp1+/- ), or no ( Spp1+/+ ) copy of the endogenous Spp1 gene. Primary end-points were lung tumour number and size; secondary end-points were SPP1 expression, epithelial cell survival, carcinogen-induced inflammation, and angiogenesis. Data are presented as mean ± SD. Compared with Spp1+/+ mice (n = 22), Spp1-/- mice (n = 25) developed dramatically fewer and significantly smaller lung tumours in response to urethane, while Spp1± mice (n = 12) behaved similar to Spp1-/- mice (number/diameter of lung tumours in Spp1+/+ , Spp1+/- , and Spp1-/- mice, respectively: 16.1 ± 12.7/1.2 ± 0.3 mm, 2.4 ± 2.3/0.9 ± 0.2 mm, and 1.3 ± 1.6/0.7 ± 0.2 mm; P Spp1+/+ with Spp1-/- and Spp1+/- mice). Spp1-/- mice were also protected from two-hit MCA/BHT-oncogenesis compared with Spp1+/+ controls. Spp1-/- mice displayed decreased epithelial cell survival and reduced numbers of airspace macrophages early after urethane, and enhanced tumour cell apoptosis and limited tumour angiogenesis at late stages of lung tumour progression. SPP1 was expressed in the naive lung by non-ciliated airway epithelial cells and alveolar macrophages and was significantly up-regulated during multi-stage lung carcinogenesis. Our data indicate that SPP1 is functionally involved in airway epithelial carcinogenesis and may present a target for lung cancer treatment and prevention.

cAMP-response element binding protein (CREB) is an essential survival factor for normal respiratory epithelium, however, its role in lung cancer development has not been investigated. Our aim was to define the impact of CREB on KRAS -driven lung adenocarcinoma. Wild-type (W), conditional mutant KRAS- expressing ( LSL.KRAS G12D ; K), and Creb1 -deleted ( Creb1 f/f ; C) mice were intercrossed in all possible combinations, followed by intraperitoneal urethane or single intratracheal adenovirus (Ad)-Cre delivery.Conditional Creb1 -deleted lung cancer cell lines (CCLC) were derived from the lungs of urethane- but not Ad- Cre -treated C mice.(Ad)-Cre-treated C mice were markedly protected from urethane-triggered lung tumorigenesis compared with W controls [lung tumor mass, median(95%CI), respectively: 1(5-11) and 24(19-45) μL; P In vitro Cre-mediated Creb1 deletion or treatment of CCLC with CREB-inhibitor ICG-001 diminished cellular proliferation, in vivo tumor growth, and spontaneous lung metastasis. KRAS silencing in CCLC lead to decreased CREB activation, whereas microarray analysis of Creb-1 deleted CCLC revealed marked reductions in C-X-C chemokine expression. CREB signaling is essential for KRAS -driven lung adenocarcinoma by augmenting tumor-associated inflammation. This work was supported by European Research Council Starting Independent Investigator (#260524) and Proof-of-Concept (#679345) Grants.

Together with brain, liver, and bones, the lungs are frequent targets of tumor metastasis. Although secreted phosphoprotein 1 (SPP1) has been associated with the dissemination of various bodily tumors, its role in lung metastasis remains unknown. To investigate the mechanism by which SPP1 induces pulmonary metastasis, we employed mouse models of spontaneous (s.c. injection of a million tumor cells) and induced (i.v. injection of half a million tumor cells) lung-targeted metastasis of three cancer cell lines (MC38 colon and LLC lung adenocarcinomas; B16F10 melanoma) in syngeneic C57BL/6 mice competent (Spp1 +/+ ) and deficient (Spp1 -/- )in Spp1 alleles. Tumor-derived SPP1 expression was modulated using stable anti-Spp1 shRNA and forced overexpression of intracellular (Spp1is2) and secreted (Spp1is4) Spp1 isoforms. We determined that lung epithelial cells expressed predominantly SPP1is4, which played no role in pulmonary metastasis, since Spp1 +/+ and Spp1 -/- mice equally developed lung metastases. On the contrary, tumor cells expressing both isoforms, exerted distinct effects: SPP1is2 promoted blood-borne tumor cell survival via suppressing TRP53 expression, whereas SPP1is4 accelerated lung metastasis by enhancing tumor-derived CCL2 secretion. Consistently with the above findings, Ccr2 -/- mice were protected from lung metastasis. In addition, SPP1-competent and incompetent tumor cells could equally colonize the lungs of both Ccr2 +/+ and Ccr2 -/- mice, indicating the role of CCL2 to SPP1 mediated effects. In conclusion, our data indicate that tumor-derived SPP1 promotes pneumotropic metastasis and presents a possible therapeutic target aimed at preventing lung tumor dissemination.

Background Carcinogen-inflicted human cancers, including lung tumours harbour thousands of mutations per genome, most of which are unknown (Garraway, LA et al, Cell 2013;153:17–37). Aim To develop a faithful mouse model of human tobacco carcinogen-induced lung adenocarcinoma suitable for the identification of novel oncogenic genes and pathways. Methods We repeatedly managed to obtain several murine lung adenocarcinoma cell lines (MLA) by chronically exposing various mouse strains to different tobacco carcinogens. MLA were characterised for cancer stemness and oncogenes, as well as global gene expression. Results To date, 12 MLA cell lines have been derived from Wt and transgenic mice on the FVB, Balb/c, and C57BL/6 strains by means of urethane or diethylnitrosamine exposure. All MLA were immortal, phenotypically stable, and indefinitely passaged in vitro over a period of over 18 months and/or 60 passages. In addition, all cell lines were oncogenic, transplantable, metastatic, and uniformly lethal in vivo. Interestingly, MLA displayed Kras mutations in codon 61, mono- or bi-allelic Trp53 loss, and expression of lung cancer stemness factors Itgb3 and Lgr6, in amazing similarity to human lung cancers. Microarray revealed that all MLA cell lines heavily overexpressed Prl2c2, encoding proliferin, in comparison to the native lungs. Prl2c2 silencing diminished MLA proliferation and stemness, to a degree comparable with Itgb3 interference. Conclusions MLA are faithful models of human lung adenocarcinoma that led to the discovery of Prl2c2 as a candidate lung tumour promoter. Funding European Research Council Starting Independent Investigator Grant #260524. Respire 2 European Respiratory Society Fellowship, European Respiratory Society Short Term Research Fellowship.

Increased expression of osteopontin (secreted phosphoprotein 1, SPP1) is associated with aggressive human lung adenocarcinoma (LADC), but its function remains unknown. Our aim was to determine the role of SPP1 in smoking-induced LADC. We combined mouse models of tobacco carcinogen-induced LADC, of deficiency of endogenous Spp1 alleles, and of adoptive pulmonary macrophage reconstitution to map the expression of SPP1 and its receptors and determine its impact during carcinogenesis. Co-expression of Spp1 and mutant KrasG12C in benign cells was employed to investigate SPP1/KRAS interactions in oncogenesis. Finally, intratracheal adenovirus encoding Cre recombinase was delivered to LSL.KRASG12D mice lacking endogenous or overexpressing transgenic Spp1 alleles. SPP1 was overexpressed in experimental and human LADC and portended poor survival. In response to two different smoke carcinogens, Spp1-deficient mice developed fewer and smaller LADC with decreased cellular survival and angiogenesis. Both lung epithelial- and macrophage-secreted SPP1 drove tumor-associated inflammation, while epithelial SPP1 promoted early tumorigenesis by fostering the survival of KRAS-mutated cells. Finally, loss and overexpression of Spp1 was, respectively, protective and deleterious for mice harboring KRASG12D-driven LADC. Our data support that SPP1 is functionally involved in early stages of airway epithelial carcinogenesis driven by smoking and mutant KRAS and may present an important therapeutic target.

Mast cells (MC) have been identified in human lung adenocarcinoma (LADC) tissues, but their functional role has not been investigated in vivo. For this, we applied three mouse models of KRAS-mutant LADC to two different MC-deficient mouse strains (cKitWsh and Cpa3.Cre). Moreover, we derived MC gene signatures from murine bone marrow-derived MC and used them to interrogate five human cohorts of LADC patients. Tumor-free cKitWsh and Cpa3.Cre mice were deficient in alveolar and skin KIT-dependent (KIT+) MC, but cKitWsh mice retained normal KIT-independent (KIT-) MC in the airways. Both KIT+ and KIT- MC infiltrated murine LADC to varying degrees, but KIT+ MC were more abundant and promoted LADC initiation and progression through interleukin-1β secretion. KIT+ MC and their transcriptional signature were significantly enriched in human LADC compared to adjacent normal tissue, especially in the subset of patients with KRAS mutations. Importantly, MC density increased with tumor stage and high overall expression of the KIT+ MC signature portended poor survival. Collectively, our results indicate that KIT+ MC foster LADC development and represent marked therapeutic targets.

Lung adenocarcinoma (LUAD) and chronic lung diseases caused by smoking and environmental noxious agents are the deadliest diseases worldwide, sharing a partially charted pathobiology of dysfunctional alveolar repair. Here we sought to identify the respiratory epithelial dynamics and molecular signatures participating in adult lung maintenance and chemical carcinogenesis. We employed novel mouse models of respiratory epithelial marking and ablation, a battery of pulmonary toxins and carcinogens, experimental protocols of carcinogen-induced LUAD, tobacco carcinogen-induced LUAD cell lines, and human transcriptomic data and identified a prominent involvement of airway molecular programs in alveolar maintenance and carcinogen-induced LUAD. The airway-specific transcriptomic signature was redistributed to the alveoli after toxic and carcinogenic insults and resulted in marked contributions of airway-labeled cells to injury-recovered alveoli and LUAD. Airway cells maintained Kras mutations and therefore possibly contributed to lung cancer initiation, while LUAD were spatially linked to neighboring airways. Transcriptomic profiling of carcinogen-induced murine and human LUAD revealed enrichment in airway signatures, while ablation of airway cells distorted alveolar structure and function and protected mice from LUAD development. Collectively, these results indicate that airway cells and/or transcriptomic signatures are essential for alveolar maintenance and LUAD development.

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB–activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D. Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of RelB, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS-mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo. IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS-mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease. Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939–51. ©2018 AACR.

Malignant pleural effusion (MPE) is a frequent metastatic manifestation of human cancers. While we previously identified KRAS mutations as molecular culprits of MPE formation, the underlying mechanism remained unknown. Here, we determine that non-canonical IKKα-RelB pathway activation of KRAS-mutant tumor cells mediates MPE development and this is fueled by host-provided interleukin IL-1β. Indeed, IKKα is required for the MPE-competence of KRAS-mutant tumor cells by activating non-canonical NF-κB signaling. IL-1β fuels addiction of mutant KRAS to IKKα resulting in increased CXCL1 secretion that fosters MPE-associated inflammation. Importantly, IL-1β-mediated NF-κB induction in KRAS-mutant tumor cells, as well as their resulting MPE-competence, can only be blocked by co-inhibition of both KRAS and IKKα, a strategy that overcomes drug resistance to individual treatments. Hence we show that mutant KRAS facilitates IKKα-mediated responsiveness of tumor cells to host IL-1β, thereby establishing a host-to-tumor signaling circuit that culminates in inflammatory MPE development and drug resistance., Malignant pleural effusion (MPE) is a life-threatening cancer-related disorder. Here, the authors show that KRAS-mutant tumor cells require IKKα, activated via host-provided IL-1β, to promote MPE development and that co-inhibition of both KRAS and IKKα ameliorates the development of MPE in mouse models.

This study aims to address the role of focal adhesion proteins α- and β-parvin in human colorectal carcinoma (CRC). Expression of α- and β-parvin was examined by immunohisto- chemistry and real-time RT-PCR in a series of human CRC. Parvins were overexpressed in CRC and their expression correlated significantly with tumor invasion, lymph node metastasis, and disease stage. A significant positive correlation of parvins protein expression with overexpression of integrin-linked kinase, p-AKT, and nuclear β-catenin, as well as with downregulation of E-cadherin was also observed. In conclusion, overexpression of α- and β-parvin seems to be implicated in human colorectal cancer progression.

An ischemia/reperfusion injury of rat’s sciatic nerve was experimentally developed. In this model, we measured the in vivo production of superoxide radical, as a marker of oxidative stress and the occludin expression as an indicator of blood-nerve barrier function and we examined potential protective innervations against these abnormalities. Right sciatic nerves of the animals underwent 3 h of ischemia followed by 7 days of reperfusion and were divided into three groups: ischemic, pretreated with vitamin C in conjunction with vitamin E and treated with tissue plasminogen activator. Compared to measurements from left sciatic nerves used as sham, the ischemic group showed significantly increased superoxide radical and reduced expression of occludin in western blot and immunohistochemistry. No such differences were detected between sham and nerves in the vitamin or tissue plasminogen activator groups. It is suggested that the experimental ischemia/reperfusion model was suitable for studying the relationship between oxidative state and blood-nerve barrier. The reversion of abnormalities by the applied neuroprotective agents might prove to be a clinically important finding in view of the implication of vascular supply derangement in various neuropathies in humans.

Background: Malignant pleural mesothelioma (MPM) is a highly aggressive tumor linked to loss of tumor suppressorsBAP1, NF2, andTRP53and accompanied by malignant pleural effusion (MPE). Aim: To develop novel mouse models of MPM with MPE. Methods: Intercrosses of conditional mutantKRAS (LoxP-STOP-LoxP.KRASG12D; K) andTRP53-deleted (LoxP.Trp53.LoxP; P) mice received intrapleural adenovirus-Cre. Pleural tumors were molecularly phenotyped and cultured to isolate cell lines, which were inoculated into the pleurae of naive mice. Results: K mice developed lethal epithelioid MPM (median, 95%CI survival: 339, 285-379 days). KP mice succumbed to sarcomatoid MPM (median, 95%CI survival 40, 38-72 days) accompanied by MPE (mean±SD volume 349±269 µL). KP mice also developed peritoneal mesothelioma upon intraperitoneal adenovirus-Cre. Mouse MPMs expressed calretinin, podoplanin, and osteopontin, but not surfactant protein C. MPM cell lines were isolated from KP MPM, which transplanted MPM to naiveC57BL/6mice causing their death (median, 95%CI survival 26, 25-28 days) by MPE induction (mean±SD volume 387±180 µL). The models harbored secondary BAP1, NF2 and CDKN2 mutations. Treatment of KP mice with a KRAS inhibitor resulted in increased survival and halted MPM growth and MPE formation. Conclusions: We developed multiple models of primary and transplantable MPM that show thatKRASandTP53mutations can initiate the disease and cause secondaryBAP1andNF2loss. Funding ERC Grants #260524 and #679345.

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition., Malignant pleural effusion (MPE) is a lethal condition associated with various cancers. Here, the authors show that cancer cells with KRAS mutations promote MPE by recruiting myeloid cells via CCL2 signalling and that pharmaceutical targeting of KRAS results in reduced MPE incidence and volume in mouse models.

Chronic lung diseases present tremendous health burdens and share a common pathobiology of dysfunctional epithelial repair. Lung adenocarcinoma, the leading cancer killer worldwide, is caused mainly by chemical carcinogens of tobacco smoke that induce mutations in pulmonary epithelial cells leading to uncontrolled epithelial proliferation. Lung epithelial cells that possess the capacity for self-renewal and regeneration of other lung cell types are believed to underlie the pathobiology of chronic obstructive, fibrotic and neoplastic lung disorders. However, the understanding of lung epithelial progenitor cell hierarchy and turnover is incomplete and a comprehensive model of the cellular and transcriptional events that underlie lung regeneration and carcinogenesis is missing. The mapping of these processes is extremely important, since their modulation would potentially allow effective cure and/or prevention of chronic lung diseases. In this review we describe current knowledge on cellular and molecular pathways at play during lung repair and carcinogenesis and summarise the critical lung cell populations with regenerative and cancerous potential.

The lungs are frequently affected by cancer metastasis. Although NRAS mutations have been associated with metastatic potential, their exact role in lung homing is incompletely understood. We cross‐examined the genotype of various tumor cells with their ability for automatic pulmonary dissemination, modulated NRAS expression using RNA interference and NRAS overexpression, identified NRAS signaling partners by microarray, and validated them using Cxcr1‐ and Cxcr2‐deficient mice. Mouse models of spontaneous lung metastasis revealed that mutant or overexpressed NRAS promotes lung colonization by regulating interleukin‐8‐related chemokine expression, thereby initiating interactions between tumor cells, the pulmonary vasculature, and myeloid cells. Our results support a model where NRAS‐mutant, chemokine‐expressing circulating tumor cells target the CXCR1‐expressing lung vasculature and recruit CXCR2‐expressing myeloid cells to initiate metastasis. We further describe a clinically relevant approach to prevent NRAS‐driven pulmonary metastasis by inhibiting chemokine signaling. In conclusion, NRAS promotes the colonization of the lungs by various tumor types in mouse models. IL‐8‐related chemokines, NRAS signaling partners in this process, may constitute an important therapeutic target against pulmonary involvement by cancers of other organs.