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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.

Human kallikrein-related peptidase 6 (KLK6) was cloned as a putative class II tumor suppressor based on its inactivated expression in metastatic breast cancer. Here, we investigated the mechanism(s) underlying the silencing of KLK6 gene in metastatic breast cancer and its putative implications for tumor progression. We present evidence that tumor-specific loss of KLK6 expression is due to hypermethylation of specific CpGs located in the KLK6 proximal promoter. Methylation-dependent binding of methyl CpG-binding protein 2 and the formation of repressive chromatin mediated by localized histone deacetylation are critical components of KLK6 silencing in breast tumors. Re-expression of KLK6 in nonexpressing MDA-MB-231 breast tumor cells by stable cDNA transfection resulted in marked reversal of their malignant phenotype, manifested by lower proliferation rates and saturation density, marked inhibition of anchorage-independent growth, reduced cell motility, and their dramatically reduced ability to form tumors when implanted in severe combined immunodeficiency mice. Interestingly, inhibition of tumor growth was observed at physiologic concentrations of KLK6, but not when KLK6 was highly overexpressed, as observed in a subset of breast tumors. Differential proteomic profiling revealed that KLK6 re-expression results in significant down-regulation of vimentin which represents an established marker of epithelial-to-mesenchymal transition of tumor cells and in concomitant up-regulation of calreticulin and epithelial markers cytokeratin 8 and 19, indicating that KLK6 may play a protective role against tumor progression that is likely mediated by inhibition of epithelial-to-mesenchymal transition. We suggest that KLK6 is an epigenetically regulated tumor suppressor in human breast cancer and provide ways of pharmacologic modulation. [Cancer Res 2009;69(9):3779–87]

Supplementary Figure 2 from A Tumor-Protective Role for Human Kallikrein-Related Peptidase 6 in Breast Cancer Mediated by Inhibition of Epithelial-to-Mesenchymal Transition

Supplementary Figure 1 from A Tumor-Protective Role for Human Kallikrein-Related Peptidase 6 in Breast Cancer Mediated by Inhibition of Epithelial-to-Mesenchymal Transition

Supplementary Figure Legends 1-2 from A Tumor-Protective Role for Human Kallikrein-Related Peptidase 6 in Breast Cancer Mediated by Inhibition of Epithelial-to-Mesenchymal Transition

Supplementary Table 1 from A Tumor-Protective Role for Human Kallikrein-Related Peptidase 6 in Breast Cancer Mediated by Inhibition of Epithelial-to-Mesenchymal Transition

Hepatocellular carcinoma (HCC) ranks among the five most common cancer entities worldwide and leads to hundred-thousands of deaths every year. Despite some groundbreaking therapeutical revelations during the last years, the overall prognosis remains poor. Although the immune system fights malignant transformations with a robust anti-tumor response, certain immune mediators have also been shown to promote cancer development. For example, interleukin (IL)-22 has been associated with HCC progression and worsened prognosis in multiple studies. However, the underlying mechanisms of the pathological role of IL-22-signaling as well as the role of its natural antagonist IL-22 binding protein (IL-22BP) in HCC remain elusive. Here, we corroborate the pathogenic role of IL-22 in HCC by taking advantage of two mouse models. Moreover, we observed a protective role of IL-22BP during liver carcinogenesis. While IL-22 was mainly produced by CD4+ T cells in HCC, IL-22BP was abundantly expressed by neutrophils during liver carcinogenesis. Hepatocytes could be identified as a major target of this pathological IL-22-signaling. Moreover, abrogation of IL-22 signaling in hepatocytes in IL22ra1flox/flox × AlbCre+ mice reduced STEAP4 expression-a known oncogene-in HCC in vivo. Likewise, STEAP4 expression correlated with IL22 levels in human HCC samples, but not in healthy liver specimens. In conclusion, these data encourage the development of therapeutical approaches that target the IL-22–IL-22BP axis in HCC.

The clearance of apoptotic cells by Mφs favors malignant pleural effusion progression in a murine cancer model., Malignant pleural effusion (MPE) results from the capacity of several human cancers to metastasize to the pleural cavity. No effective treatments are currently available, reflecting our insufficient understanding of the basic mechanisms leading to MPE progression. Here, we found that efferocytosis through the receptor tyrosine kinases AXL and MERTK led to the production of interleukin-10 (IL-10) by four distinct pleural cavity macrophage (Mφ) subpopulations characterized by different metabolic states and cell chemotaxis properties. In turn, IL-10 acts on dendritic cells (DCs) inducing the production of tissue inhibitor of metalloproteinases 1 (TIMP1). Genetic ablation of Axl and Mertk in Mφs or IL-10 receptor in DCs or Timp1 substantially reduced MPE progression. Our results delineate an inflammatory cascade—from the clearance of apoptotic cells by Mφs, to production of IL-10, to induction of TIMP1 in DCs—that facilitates MPE progression. This inflammatory cascade offers a series of therapeutic targets for MPE.

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.

Acute liver injury can be secondary to a variety of causes, including infections, intoxication, and ischemia. All of these insults induce hepatocyte death and subsequent inflammation, which can make acute liver injury a life-threatening event. IL-22 is a dual natured cytokine which has context-dependent protective and pathogenic properties during tissue damage. Accordingly, IL-22 was shown to promote liver regeneration upon acute liver damage. However, other studies suggest pathogenic properties of IL-22 during chronic liver injury. IL-22 binding protein (IL-22BP, IL-22Ra2) is a soluble inhibitor of IL-22 that regulates IL-22 activity. However, the significance of endogenous IL-22BP in acute liver injury is unknown. We hypothesized that IL-22BP may play a role in acute liver injury. To test this hypothesis, we used Il22bp-deficient mice and murine models of acute liver damage induced by ischemia reperfusion and N-acetyl-p-aminophenol (acetaminophen) administration. We found that Il22bp-deficient mice were more susceptible to acute liver damage in both models. We used Il22 × Il22bp double-deficient mice to show that this effect is indeed due to uncontrolled IL-22 activity. We could demonstrate mechanistically increased expression of Cxcl10 by hepatocytes, and consequently increased infiltration of inflammatory CD11b+Ly6C+ monocytes into the liver in Il22bp-deficient mice upon liver damage. Accordingly, neutralization of CXCL10 reversed the increased disease susceptibility of Il22bp-deficient mice. In conclusion, our data indicate that IL-22BP plays a protective role in acute liver damage, via controlling IL-22–induced Cxcl10 expression.

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TH17 cells exemplify environmental immune adaptation: they can acquire both a pathogenic and an anti-inflammatory fate. However, it is not known whether the anti-inflammatory fate is merely a vestigial trait, or whether it serves to preserve the integrity of the host tissues. Here we show that the capacity of TH17 cells to acquire an anti-inflammatory fate is necessary to sustain immunological tolerance, yet it impairs immune protection against S. aureus. Additionally, we find that TGF-β signalling via Smad3/Smad4 is sufficient for the expression of the anti-inflammatory cytokine, IL-10, in TH17 cells. Our data thus indicate a key function of TH17 cell plasticity in maintaining immune homeostasis, and dissect the molecular mechanisms explaining the functional flexibility of TH17 cells with regard to environmental changes., CD4+ T helper cells producing IL-17A (TH17 cells) can take on pathogenic or anti-inflammatory functions in context-specific manners. Here the authors show that the anti-inflammatory fate of TH17 cells contributes, via TGF-β signaling and induction of IL-10, to host immune tolerance, but also simultaneously dampens protective immunity against S. aureus.

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.

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.

Introduction Malignant pleural effusion (MPE) is primarily an immune-mediated manifestation of pleural-metastasized cancers, particularly adenocarcinomas that harbour KRAS mutations. We have previously determined that NF-κB activation in pleural tumor cells drives MPE formation, but the NF-kB pathways involved are obscure. Aims and objectives To investigate the relationship between mutant KRAS presence and NF-kB activation in pleural tumor cells that leads to MPE development. Methods We profiled baseline and host-microenvironment-induced NF-κB activity of tumor cells, and examined its relationship with KRAS status. We further silenced NF-kB activating kinases in tumor cells aiming to eliminate NF-kB activity and thus MPE competence. Results We found that mutant KRAS tumor cells were MPE competent and displayed IL-1b-induced IKKa nuclear translocation that was lost when KRAS was silenced. In contrast, wt KRAS cells did not form MPE and did not respond to IL-1b with nuclear IKKa shuttling. IKKa, but not IKKb, silencing of mutant KRAS tumors hampered their ability for MPE formation, suggesting a mutant KRAS -IKKa addiction in the pleural tumor cell during MPE development. Furthermore, IL-1b, but not TNFa, originated from host immune cells was required to sustain the MPE-competent phenotype of IKKa-addicted KRAS -mutant tumor cells. Microarray data showed that these cells secrete CXCL5 and CCl2 in response to IL-1b. Conclusions We demonstrate that KRAS -mutant tumor cells require host IL-1b signaling for MPE precipitation. This phenotype is mediated by IKKa. Acknowledgements These studies were supported by a European Research Council Starting Independent Investigator Grant (#260524). This abstract has been presented previously at the European Respiratory Society9s Lung Science Conference in March 2016 .

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.

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.

Upon interaction with dendritic cells (DCs), naive CD4 T cells differentiate into distinct subsets and orchestrate the development of a physiological immune response. When uncontrolled by cellular and molecular mechanisms, CD4 T cells can also lead to immune mediated inflammatory diseases (IMIDs). Initially, these distinct CD4 T-cell subsets were defined according to the expression of a limited number of cytokines. Later it was revealed that CD4 T cells can acquire much more complex functional phenotypes than previously thought. Experimental data showed that the CD4 T-cell subset TH17 can secrete IFN-γ and IL-4, which are signature molecules of other T-cell subsets. Furthermore, some TH17 cells can also explore an anti-inflammatory fate and participate in the resolution of the immune response. A more flexible theory has therefore evolved with the scope to better represent the plastic biology of CD4 T cells. In this context, several aspects still remain unclear. The goal of this review is to discuss the role of extrinsic and intrinsic cellular and molecular mechanisms, which can drive the plasticity of TH17 cells. In particular, we will outline the role of DCs and the function of transcriptional factors in shaping the fate of TH17 cells towards either a pathogenic or a regulatory phenotype. Finally, we will discuss whether TH17 cell plasticity could be a target for new therapies for IMIDs. We indeed envision that when the cellular and molecular mechanisms controlling TH17 plasticity are known, new therapies, which aim to reset the immune system, will be developed. This will be achieved by either selectively depleting only the pathogenic TH17 cells or, if possible, re converting these cells from pathogenic to regulatory. This will overcome the challenge posed by the immune suppressive side effects caused by the current therapies, which impair the function of CD4 cells or delete all of them, to the detriment of the patient.

Upon interaction with dendritic cells (DCs), naïve CD4 T cells differentiate into distinct subsets and orchestrate the development of a physiological immune response. When uncontrolled by cellular and molecular mechanisms, CD4 T cells can also lead to immune mediated inflammatory diseases (IMIDs). Initially, these distinct CD4 T-cell subsets were defined according to the expression of a limited number of cytokines. Later it was revealed that CD4 T cells can acquire much more complex functional phenotypes than previously thought. Experimental data showed that the CD4 T-cell subset T

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.

Background Nuclear factor (NF)-kB activation is essential for KRAS-mutant lung adenocarcinoma (Stathopoulos, G.T. et al. Proc Natl Acad Sci U S A 2007;104:18514-9 and Meylan, E. et al. Nature 2009;462:104-7). However, the relative contributions of NF-kB-activating kinases IKKa and IKKb are unknown. Aims To monitor and block NF-kB activation and IKK functions in lung adenocarcinoma. Methods NF-kB reporter mice and conditional IKKa/b gene deleted mice were used, which, respectively, express firefly luciferase in all somatic cells upon NF-kB activation, or lose the respective IKK gene upon CRE-mediated recombination. Lung adenocarcinomas were induced in these mice via ip injection of urethane (1 g/Kg) that is a tobacco carcinogen. Mutant KRAS-IKK interactions were studied using transfected benign HEK293T cells with mutant KRAS-and/or IKK-expression vectors. IKK-inhibitors are used in ADCA cell lines and KRAS-induced mice. Results Urethane caused strong NF-kB activation in the lungs, characterized by significant induction and nuclear translocation of IKKa, but not IKKb. Deletion of IKKa from the respiratory epithelium resulted in almost complete eradication of urethane-induced KRAS-mutant lung tumors, while deletion of IKKb was less protective. IKKb, but not IKKa, functioned as a tumor promoter in KRAS-wild-type HEK293T cells. However, only IKKa, and not IKKb, could render mutant KRAS-expressing HEK293T cells oncogenic in immunocompromized mice. In KRAS-IKKa-addicted tumorigenic HEK293T cells, IKKa resided in the nucleus.Dual IKK inhibitor is more effective in our models than IKKb inhibitor. Conclusions IKKa is required for KRAS-mediated lung adenocarcinoma. Funding European Research Council Starting Independent Investigator Grant #260524. This abstract has been presented previously at the European Respiratory Society9s Lung Science Conference in March 2016 .

Aim: To evaluate the association between the ILK-PINCH-PARVIN complex/ Rsu-1 and non-small cell lung cancer (NSCLC) and discuss its clinicopathological significance. Objective: The integrin-linked kinase (ILK)-PINCH-parvin (IPP) complex at integrin adhesion sites is a critical regulator of cell migration, invasion and metastasis. Deregulation of the IPP complex has been implicated in human carcinogenesis. Recent observations suggest that RSU-1, is a PINCH-binding partner that regulates PINCH mediated adhesion and migration. Materials and methods: Protein expression of ILK, PINCH, alpha-parvin, beta-parvin and RSU-1 in relation to clinicopathological parameters was evaluated by immunohistochemistry in 82 FFPE tissue samples of non-small cell lung cancer (NSCLC).PINCH and RSU-1 expression was also measured by Western blot analysis in lung cancer cell lines and on extracts of non-tumorigenic lungs from mice. Results: All components of the IPP complex were overexpressed while RSU-1 was downregulated in lung cancer cells compared to non-neoplastic lung parenchyma. ILK and alpha-parvin expression was significantly higher in high grade (p=0.002) and high pT (p=0.047) tumors respectively. Expression of PINCH associated significantly with lymph node metastasis (p=0.045). Subsequent in vitro experiments revealed that PINCH expression was significantly higher while RSU-1 expression decreased in the cell lines examined compared to extracts from non-tumorigenic lungs. Conclusion: Our results suggest that overexpression of the IPP complex and downregulation of RSU-1 may be implicated in lung carcinogenesis.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor caused by asbestos. Faithful mouse models of the disease are missing. Our aim was to develop and characterize a conditional and transplantable mouse model of MPM. Conditional mutant KRAS -expressing ( LSL.KRAS G12D ; K) and TRP53 -deleted ( Trp53 f/f ; P) mice were intercrossed in all possible combinations, followed by intrapleural adenovirus-Cre. Efficient and widespread pleural recombination was verified using dual fluorescent Cre-reporter mice. Multiple cell lines were derived from primary MPM from KP mice and transplanted to C57BL/6 mice. KP mice developed multifocal lethal (median, 95%CI survival 40, 38-72 days) MPM with malignant pleural effusions (MPE; mean±SD volume 349±269 μL) involving both the visceral and parietal pleura and the mediastinum. KP-MPM expresses classical markers of human MPM and gives rise to multiple MPM cell lines (KPM). KPM are transplantable to C57BL/6 mice, leading to death (median, 95%CI survival 26, 25-28 days) and MPE (mean±SD volume 387±180 μL). Δ KRAS could be the tumor initiating gene of malignant pleural mesothelioma development and could present an important therapeutic target. European Research Council Starting Independent Investigator (#260524) and Proof-of-Concept (#679345) Grants.

Osteopontin (SPP1) expression has been identified in human lung cancer and has been linked with enhanced tumor progression. To examine its functional role, we induced lung tumors 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 tumor 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 tumors in response to urethane, while Spp1+/- mice (n=12) behaved similar to Spp1-/- mice (number/diameter of lung tumors 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 tumor cell apoptosis and limited tumor angiogenesis at late stages of lung tumor 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. SPP1 also initiated the survival of mutant Kras cells. Our data indicate that SPP1 is functionally involved in airway epithelial carcinogenesis via survival of mutant Kras cells and may present a target for lung cancer treatment and prevention.

Background: The immune cells that facilitate malignant pleural effusion (MPE), an immune-mediated end-stage cancer manifestation (Stathopoulos, G.T. et al. Am J Respir Crit Care Med 2012;186:487-92), have not been identified. Aims: We found mast cells (MC) in MPE and investigated their functions. Methods: Human and experimental MPE were assessed for MC content.MPE was induced by intrapleurally injected Lewis lung (LLC) and MC38 colon adenocarcinoma cells in Wt and two separate strains of MC-deficient ( cKitWsh, Cpa3.Cre ) mice. Bone marrow-derived MC were used for MC cell fate and restoration studies. Tumor cells and tumor-exposed MC were subjected to transcriptional profiling. Results: We discovered and characterized MC in human and murine MPE. Cell fate models conveyed that MC were attracted to MPE by tumor-derived CCL2 to be locally degranulated by osteopontin. Both cKitWsh and Cpa3 . Cre mice were protected from MPE induced by both LLC and MC38 cells (mean±SD MPE volume of C57BL/6, cKitWsh and Cpa3 . Cre mice induced by LLC cells: 573±242, 108±130, and 277±284µL; and by MC38 cells: 864±310, 278±323, and 464±203 µL; n = 7-23/group; P Conclusions: Adenocarcinomas instate circuitry with MC to induce MPE once metastatic in the pleural space. Acknowledgements: Supported by ERC-2010-StG-#260524 anda Hellenic Thoracic Society 2012 Research Award.

Background: The reasons why the lungs are preferential metastatic targets for some human cancers are unknown. Aims: To unravel the mechanism of tumor pneumotropism. Methods: Several mouse and human tumor cell lines were genotyped for key oncogenes and tumor suppressors. Their potential for spontaneous lung metastasis was assessed by subcutaneous inoculation into receptive host mice. Mutant oncogenes were silenced via shRNA or introduced using relevant expression vectors. Chemokine signaling was studied using microarray, qPCR, ELISA and immunobloting and was abrogated using chemokine receptor-deficient mice. Results: Spontaneous lung metastasis co-segregated with the presence of NRAS mutations detected in 3 of 11 cell lines studied. The expected frequency of NRAS mutations based on COSMIC (http://cancer.sanger.ac.uk/cosmic/) was tightly correlated (P=0.0000054, R 2 =0.652) with lung metastasis at necropsy in 3827 patients with various cancers (Disibio, G. et al. Arch Pathol Lab Med 2008;132:931–9). Mutant NRAS was required and sufficient for lung colonization since mutant NRAS silencing and overexpression inhibited and installed tumor pneumotropism, respectively. NRAS mutations functioned to up-regulate Cxcl5/Ppbp and CXCL6/7/8 expression by mouse and human tumor cells, respectively, directly targeting circulating tumor cells to the CXCR1+ lung vasculature. Lung-homed tumor cells secondarily recruited CXCR2+ myeloid cells to the lungs. Chemokine signaling was essential for NRAS -dictated lung metastasis, since Cxcr1 and Cxcr2 deficient mice were immune to lung colonization. Conclusions: Mutant NRAS drives lung metastasis via CXCR1/2-mediated signaling events. Funding: European Research Council Starting Independent Investigator Grant #260524.

Introduction: Malignant pleural effusion (MPE) is primarily an immune-mediated manifestation of pleural-metastasized cancers, particularly adenocarcinomas that harbour KRAS mutations ( Δ KRAS ).We have previously determined that NF-κΒ activation in pleural tumor cells drives MPE formation, but the NF-κΒ pathways involved are obscure. Aims and objectives: To investigate the mechanisms of NF-κB activation in pleural tumor cells that leads to MPE development. Methods: We profiled baseline and host-induced NF-κB activity of mouse and human tumor cells, and examined its relationship with KRAS status. We further silenced NF-κB activating kinases aiming to eliminate endogenous and inducible NF-κB activity of mouse and human tumors. Results: Tumor cells bearing KRAS mutations, competent of causing MPE, displayed IL-1β-induced IKKα translocation to the nucleus that was lost when KRAS knockdown was performed. In contrast, cell lines with wt KRAS that do not cause MPE did not respond to IL-1β with nuclear IKKα shuttling. shRNA IKKα, but not IKKβ, silencing of ΔKRAS tumors severely hampered their ability for MPE formation. IKKαoverexpression in wt KRAS tumors failed to restore the MPE-competent phenotype of ΔKRAS tumors, suggesting an addiction of IKKα with Δ KRAS in the tumor cell during MPE development. Furthermore, IL-1β, but not TNFα, originated from host immune cells was required to sustain the MPE-competent phenotype of IKKα-addicted KRAS-mutant tumors. Conclusions: We demonstrate that KRAS- mutant tumor cells require host-IL-1β signaling for MPE precipitation. This phenotype is mediated by IKKα and not IKKβ. This study was supported by a European Research Council Starting Independent Investigator Grant (#260524).

Background: EGFR/KRAS axis mutations are frequent in human malignantpleuraleffusions (MPE). Aims: To investigate the relationship between perturbations in the EGFR-KRAS axis and malignant pleural effusion development. Methods: Several mouse and human cancer cell lines were characterized for EGFR/KRAS status and for their MPE competence in appropriate hosts. EGFR/KRAS signalling was modulated in various cell lines via standard molecular techniques. MPE-associated inflammation was monitored by bioluminescence imaging, immunophenotyped by flow cytometry, and modulated by splenectomy and adoptive bone marrow transfer. EGFR/KRAS axis was targeted using gefitinib, deltarasin, and anti-CCL2 antibodies. Results: A causative relationship between Δ Kras and MPEwas established by showing that mouse and human cancer cell lines bearing Δ Kras are MPE competent and that activated Kras is capable of conferring MPE competency to human and mouse cells. Furthermore, Kras dependent downstream paracrine signaling to bone marrow activated a distinct GR1+/CD11B+ myeloid cell population, which is maturing in the spleen and is consequently recruited to the pleural cavity in a cancer-elaborated CCL2-dependent way. Splenectomy partially protects hosts from MPE. The pharmacological manipulation of Δ EGFR/ Δ KRAS axis with Gefitinib (targeting Δ EGFR ) or Deltarasin (targeting Δ KRAS ) and the antibody-mediated neutralization of CCL2 completely annihilate MPE formation and the recruitment of CD11b+/GR1+ cells to the pleura. Conclusions: Our work provides insights to MPE pathobiology and a multitude of strategies to target a currently untreatable terminal human condition. Funding: European Research Council Starting Independent Investigator Grant #260524.

Background: Carcinogen-inflicted human cancers, including lung tumors harbor thousands of mutations per genome, most of which are unknown (Garraway, L.A. 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 characterized 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 tumor promoter. Funding: European Research Council Starting Independent Investigator Grant #260524.

We report the results of experiments designed to test the histone code hypothesis. We found that only a small subset of lysines in histones H4 and H3 are acetylated in vivo by the GCN5 acetyltransferase during activation of the IFN-β gene. Reconstitution of recombinant nucleosomes bearing mutations in these lysine residues revealed the cascade of gene activation via a point-by-point interpretation of the histone code through the ordered recruitment of bromodomain-containing transcription complexes. Acetylation of histone H4 K8 mediates recruitment of the SWI/SNF complex whereas acetylation of K9 and K14 in histone H3 is critical for the recruitment of TFIID. Thus, the information contained in the DNA address of the enhancer is transferred to the histone N termini by generating novel adhesive surfaces required for the recruitment of transcription complexes.

We show that the IRF-2 oncoprotein represses virus-induced IFN-beta gene transcription via a novel mechanism. Virus infection induces recruitment of IRF-2 to some of the endogenous IFN-beta enhancers as part of the enhanceosome. Enhanceosomes bearing IRF-2 cannot activate transcription, due to the presence of a domain in IRF-2 that prevents enhanceosome-dependent recruitment of the CBP-Pol II holoenzyme complex. As a consequence, IRF-2 incorporation into enhanceosomes restricts the number of IFN-beta promoters directing transcription. Remarkably, deletion of the IRF-2 gene increases IFN-beta expression by expanding the number of cells capable of inducing IFN-beta gene transcription in response to virus infection.

The lungs are ubiquitous receptacles of metastases originating from various bodily tumors. Although osteopontin (SPP1) has been associated with tumor dissemination, the role of its isoforms in lung-directed metastasis is incompletely understood. We employed syngeneic mouse models of spontaneous and induced lung-targeted metastasis in C57BL/6 mice competent and deficient in both Spp1 alleles. Tumor-derived osteopontin expression was modulated using either stable anti-Spp1 RNA interference, or forced overexpression of intracellular and secreted Spp1 isoforms. Identified osteopontin's downstream partners were validated using lung adenocarcinoma cells conditionally lacking the Trp53 gene and Ccr2-deficient mice. We determined that host-derived osteopontin was dispensable for pulmonary colonization by different tumor types. Oppositely, tumor-originated intracellular osteopontin promoted tumor cell survival by preventing tumor-related protein 53-mediated apoptosis, while the secretory osteopontin functioned in a paracrine mode to accelerate lung metastasis by enhancing tumor-derived C-C-motif chemokine ligand 2 signaling to cognate host receptors. As new ways to target osteopontin signaling are becoming available, the cytokine may constitute an important therapeutic target against pulmonary involvement by cancers of other organs.

The 5-methyl cytosine (5meC) genomic methylation patterns play crucial roles in mammalian development and are altered in cancer. The enzymes that create, maintain and modify the DNA methylation patterns are the DNA methyltransferases (Dnmts) which are all encoded by essential genes. The de novo Dnmts -Dnmt3a and Dnmt3b- establish the DNA methylation patterns early in mammalian development by introducing DNA methylation marks where no previous methylation exists. These enzymes do not exhibit affinity for specific DNA sequences, thus their recruitment to specific DNA loci and their activities must be tightly regulated. In particular, Dnmt3a2 -one of the two protein isoforms produced by the Dnmt3a locus- is the most abundant DNA methyltransferase in mouse Embryonic Stem Cells. To identify Dnmt3a (and DNA methylation) regulators we have searched for Dnmt3a2 interacting proteins in mESCs by pull down and Mass Spectrometry. The DEAD box p68/Ddx5 RNA helicase was identified to directly interact with Dnmt3a1 and Dnmt3a2 in vitro and in vivo. We have created a mutant Ddx5 (Ddx5 MUT ) protein exhibiting an altered nuclear localization pattern compared to the wt protein. Both wt and mutant Ddx5 interact directly in vitro and co localize in vivo with Dnmt3a proteins. Our data suggest that the Dnmt3a/Ddx5 interaction might be significant for modulating the DNA methylation/demethylation dynamics in vivo.

Dnmt3a1 and Dnmt3a2 are two de novo DNA methyltransferases expressed in mouse embryonic stem cells (mESCs). They differ in that a 219-amino-acid (aa) amino (N)-terminal noncatalytic domain is present only in Dnmt3a1. Here, we examined the unique functions of Dnmt3a1 in mESCs by targeting the coding sequence of the Dnmt3a1 N-terminal domain tagged with enhanced green fluorescent protein (GFP) for insertion into the mouse Rosa26 locus. Using these targeted cells (GFP-3a1Nter), we showed that Dnmt3a1 was efficiently recruited to the silenced Oct3/4 and activated Vtn (vitronectin) gene promoters via its unique N-terminal domain. This recruitment affected the two genes in contrasting ways, compromising Oct3/4 gene promoter DNA methylation to prevent consolidation of the silent state while significantly reducing Vtn transcription. We used this negative effect of the Dnmt3a1 N-terminal domain to investigate the extent of transcriptional regulation by Dnmt3a1 in mESCs by using microarrays. A small group of all-trans retinoic acid (tRA)-inducible genes had lower transcript levels in GFP-3a1Nter cells than in wild-type mESCs. Intriguingly, this group included genes that are important for fetal nutrition, placenta development, and metabolic functions and is enriched for a distinct set of imprinted genes. We also identified a larger group of genes that showed higher transcript levels in the GFP-3a1Nter-expressing cells than in wild-type mESCs, including pluripotency factors and key regulators of primordial germ cell differentiation. Thus, Dnmt3a1 in mESCs functions primarily as a negative and to a lesser extent as a positive regulator of transcription. Our findings suggest that Dnmt3a1 positively affects transcription of specific genes at the promoter level and targets chromosomal domains to epigenetically silence gene clusters in mESCs.