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ATX staining in two mouse lung cancer models

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).

Pathogenesis and progression of lung cancer are governed by complex interactions between the environment and host genetic susceptibility, which is further modulated by genetic and epigenetic changes. Autotaxin (ATX, ENPP2) is a secreted glycoprotein that catalyzes the extracellular production of lysophosphatidic acid (LPA), a growth-factor–like phospholipid that is further regulated by phospholipid phosphatases (PLPP). LPA's pleiotropic effects in almost all cell types are mediated through at least six G-protein coupled LPA receptors (LPAR) that exhibit overlapping specificities, widespread distribution, and differential expression profiles. Here we use both preclinical models of lung cancer and clinical samples (from patients and healthy controls) to investigate the expression levels, activity, and biological role of the above components of the ATX/LPA axis in lung cancer. ENPP2 was genetically altered in 8% of patients with lung cancer, whereas increased ATX staining and activity were detected in patient biopsies and sera, respectively. Moreover, PLPP3 expression was consistently downregulated in patients with lung cancer. Comparable observations were made in the two most widely used animal models of lung cancer, the carcinogen urethane–induced and the genetically engineered K-rasG12D–driven models, where genetic deletion of Enpp2 or Lpar1 resulted in disease attenuation, thus confirming a procarcinogenic role of LPA signaling in the lung. Expression profiling data analysis suggested that metabolic rewiring may be implicated in the procarcinogenic effects of the ATX/LPA axis in K-ras-G12D–driven lung cancer pathogenesis.Significance: These findings establish the role of ATX/LPA in lung carcinogenesis, thus expanding the mechanistic links between pulmonary fibrosis and cancer. Cancer Res; 78(13); 3634–44. ©2018 AACR.

Clinicopathological characteristics of LC patients with serum samples

Supplementary Materials and Methods

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.

Tumor necrosis factor (TNF)-α is present in the microenvironment of human tumors, including malignant pleural effusion (MPE). Although the cytokine is produced in the pleural cavity by both tumor and host cells, its effects on MPE formation are unknown. In these studies, we sought to determine the role of TNF-α in the pathogenesis of MPE and to assess the therapeutic effects of its neutralization in a preclinical model. For this, MPEs were generated in immunocompetent mice using intrapleural injection of mouse lung adenocarcinoma cells. The roles of tumor- and host-derived TNF-α were assessed using combined experimentation with TNF-α gene–deficient mice and in vivo TNF-α neutralization. To expand the scope of preclinical data, TNF-α and vascular endothelial growth factor (VEGF) expression were determined in human cancer cell lines and human MPE. In the MPE model, TNF-α of host and tumor origin was present. TNF-α neutralization significantly limited tumor dissemination, effusion formation, vascular hyperpermeability, TNF-α and VEGF expression, and angiogenesis, thereby improving survival. In contrast, these variables were not different between TNF-α gene–sufficient and TNF-α gene–deficient mice. In mouse cancer cells, TNF-α functioned via nuclear factor-κB– and neutral sphingomyelinase–dependent pathways to induce TNF-α and VEGF, respectively. These results were recapitulated in human cancer cells, and a correlation was detected between TNF-α and VEGF content of human MPE. We conclude that tumor-derived TNF-α is important in the development of MPE in mice, and provide preclinical evidence supporting the efficacy of TNF-α blockade against malignant pleural disease. [Cancer Res 2007;67(20):9825–34]

Supplementary Figure Legend from Tumor Necrosis Factor-α Promotes Malignant Pleural Effusion

Figure S2. Deficiency in IL-5 does not affect infiltration of lungs with T and B lymphocytes and NK cells during initial stages of metastatic load of LLC cells.

Supplementary Figure 1 from Tumor Necrosis Factor-α Promotes Malignant Pleural Effusion

Kirsten rat sarcoma virus (KRAS)-mutant cancers are frequent, metastatic, lethal, and largely undruggable. While interleukin (IL)-1β and nuclear factor (NF)-κB inhibition hold promise against cancer, untargeted treatments are not effective. Here, we show that human KRAS-mutant cancers are addicted to IL-1β via inflammatory versican signaling to macrophage inhibitor of NF-κB kinase (IKK) β. Human pan-cancer and experimental NF-κB reporter, transcriptome, and proteome screens reveal that KRAS-mutant tumors trigger macrophage IKKβ activation and IL-1β release via secretory versican. Tumor-specific versican silencing and macrophage-restricted IKKβ deletion prevents myeloid NF-κB activation and metastasis. Versican and IKKβ are mutually addicted and/or overexpressed in human cancers and possess diagnostic and prognostic power. Non-oncogene KRAS/IL-1β addiction is abolished by IL-1β and TLR1/2 inhibition, indicating cardinal and actionable roles for versican and IKKβ in metastasis.

Summary Conventional chemotherapy leads to severe adverse effects since it involves systemic administration of toxic drugs at high dosage. Unlike traditional chemotherapeutics, calcium phosphate and citrate have both been discussed as very promising anticancer agents and are not inherently toxic. Yet, their breakthrough has been hampered by the lack of an administration approach that overcomes the strict regulatory mechanisms of the cell. Here, we present a combinatorial administration of calcium, phosphate, and citrate as colloidal, amorphous nanoparticles (CPCs) that selectively kill cancer cells without involvement of inherently toxic drugs. The particles are toxic neither before endosomal release nor after their degradation. This highly selective toxicity allowed us to successfully treat two different aggressive pleural tumors in mice, reducing their size by about 40% and 70% after only two local applications. Safety assessment studies over 2 months show no signs of adverse effects except for slightly enhanced pleural thickening after eight applications.

PURPOSE: In radiation risk analysis the state-of-the-art approach is based on descriptive models which link excess rates of cancer incidence and mortality to radiation exposure by statistical association. To estimate the number of sporadic and radiation-induced cases descriptive models apply parametric dose response function which directly determine the radiation risk. In biologically-based models of cancer risk (BBCR models) dose responses are implemented for key events on the biological level such as early mutations or clonal expansion of initiated cells. Influenced by radiation these events then shape the risk response on the epidemiological level. Although BBCR models facilitate a more comprehensive consideration of biological processes for risk assessment, their range of application in radiation research remains limited. Therefore, we emphasize their ability to improve understanding of radiation-related carcinogenesis by integrating molecular biology with epidemiology. We highlight the potential of BBCR models to harness information from adverse outcome pathways (AOPs) for risk estimation with closer links to radiobiology. The AOP concept originates from toxicology and may be applied profitably in radiation research. CONCLUSION: The conceptual design of BBCR models can be guided by the high-dimensional data environment provided by AOPs. Risk estimates from BBCR models pertain not only to classical radioepidemiological covariables such as radiation dose or attained age but also to well characterized molecular pathways. By additionally deploying biological information BBCR models facilitate finer risk stratification for a more personalized risk assessment. They leave behind the one-size-fits-all approach of descriptive modeling with the downside of more involved model development. Importantly, predictions from BBCR models can be validated against molecular measurements. Validated predictions would confirm the model design and strengthen the link between molecular biology and epidemiology. But the availability of cancer tissue in good quality from patients with known radiation exposure constitutes a major bottleneck. More ambitious initiative is needed to recover stored tissue samples and make them available for molecular investigations. To conclude, risk estimation will not only on rely on statistical association but will be quantitatively informed with radiobiological insight. Combined with the AOP framework BBCR models could improve accuracy and reduce uncertainty of radiation risk estimates in future research.

Integrin-linked kinase (ILK) forms a heterotrimeric protein complex with PINCH and PARVIN (IPP) in Focal Adhesions (FAs) that acts as a signaling platform between the cell and its microenvironment regulating important cancer-related functions. We aimed to elucidate the role of ILK in lung adenocarcinoma (LUADC) focusing on a possible link with KRAS oncogene. We used immunohistochemistry on human tissue samples and KRAS-driven LUADC in mice, analysis of large scale publicly available RNA sequencing data, ILK overexpression and pharmacological inhibition as well as knockdown of KRAS in lung cancer cells. ILK, PINCH1 and PARVB (IPP) proteins are overexpressed in human LUADC and KRAS-driven LUADC in mice representing poor prognostic indicators. Genes implicated in ILK signaling are significantly enriched in KRAS-driven LUADC. Silencing of KRAS, as well as, overexpression and pharmacological inhibition of ILK in lung cancer cells provide evidence of a two-way association between ILK and KRAS. Upregulation of PINCH, PARVB and Ras suppressor-1 (RSU1) expression was demonstrated in ILK overexpressing lung cancer cells in addition to a significant positive correlation between these factors in tissue samples, while KRAS silencing downregulates IPP and RSU1. Pharmacological inhibition of ILK in KRAS mutant lung cancer cells suppresses cell growth, migration, EMT and increases sensitivity to platinum-based chemotherapy. ILK promotes an aggressive lung cancer phenotype with prognostic and therapeutic value through functions that involve KRAS, IPP complex and RSU1, rendering ILK a promising biomarker and therapeutic target in lung adenocarcinoma.

The high plasticity of lung epithelial cells, has for many years, confounded the correct identification of the cell-of-origin of lung adenocarcinoma (LUAD), one of the deadliest malignancies worldwide. Here, we employ lineage-tracing mouse models to investigate the cell of origin of Eml4-Alk LUAD, and show that Club and Alveolar type 2 (AT2) cells give rise to tumours. We focus on Club cell originated tumours and find that Club cells experience an epigenetic switch by which they lose their lineage fidelity and gain an AT2-like phenotype after oncogenic transformation. Single-cell transcriptomic analyses identified two trajectories of Club cell evolution which are similar to the ones used during lung regeneration, suggesting that lung epithelial cells leverage on their plasticity and intrinsic regeneration mechanisms to give rise to a tumour. Together, this study highlights the role of Club cells in LUAD initiation, identifies the mechanism of Club cell lineage infidelity, confirms the presence of these features in human tumours, and unveils key mechanisms conferring LUAD heterogeneity.

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.

International audience; Epidemiological studies established an association between chronic inflammation and higher risk of cancer. Inhibition of proteolytic enzymes represents a potential treatment strategy for cancer and prevention of cancer metastasis. Cathepsin C (CatC) is a highly conserved lysosomal cysteine dipeptidyl aminopeptidase required for the activation of pro-inflammatory neutrophil serine proteases (NSPs, elastase, proteinase 3, cathepsin G and NSP-4). NSPs are locally released by activated neutrophils in response to pathogens and non-infectious danger signals. Activated neutrophils also release neutrophil extracellular traps (NETs) that are decorated with several neutrophil proteins, including NSPs. NSPs are not only NETs constituents but also play a role in NET formation and release. Although immune cells harbor large amounts of CatC, additional cell sources for this protease exists. Upregulation of CatC expression was observed in different tissues during carcinogenesis and correlated with metastasis and poor patient survival. Recent mechanistic studies indicated an important interaction of tumor-associated CatC, NSPs, and NETs in cancer development and metastasis and suggested CatC as a therapeutic target in a several cancer types. Cancer cell-derived CatC promotes neutrophil recruitment in the inflammatory tumor microenvironment. Because the clinical consequences of genetic CatC deficiency in humans resulting in the elimination of NSPs are mild, small molecule inhibitors of CatC are assumed as safe drugs to reduce the NSP burden. Brensocatib, a nitrile CatC inhibitor is currently tested in a phase 3 clinical trial as a novel anti-inflammatory therapy for patients with bronchiectasis. However, recently developed CatC inhibitors possibly have protective effects beyond inflammation. In this review, we describe the pathophysiological function of CatC and discuss molecular mechanisms substantiating pharmacological CatC inhibition as a potential strategy for cancer treatment.

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.

Background and objective Anti-angiogenic treatment has been recently shown to be clinically beneficial for mesothelioma patients. Angiopoietins-1 and -2 are key regulators of tumor angiogenesis. Ang-1 is mainly known to promote angiogenesis and vessel stability, while Ang-2 could serve as an antagonist of Ang-1 causing vessel regression and destabilization or enhance angiogenesis in a context-dependent manner. We hypothesized that Ang-1 would promote and Ang2 would halt experimental mesothelioma by affecting tumor angiogenesis. Methods To examine the effects of angiopoietins in mesothelioma angiogenesis and in vivo growth we constructed Ang-1 or Ang-2 overexpressing AE17 and AB1 mesothelioma cells and implanted them in the respective syngeneic animals. We also explored the clinical relevance of our observations using the human tumoral mRNAseq data available in the TCGA database. Results and conclusions Ang-1 promotes mesothelioma angiogenesis and growth while the effect of Ang-2 is context-dependent. Low Ang-1 levels in human mesotheliomas are associated with the epitheloid subtype. Tumors of high Ang-1, or concurrent high Ang-2 and VEGF expression present high PECAM-1 and CDH5 expression, markers of vascularity and vascular stability, respectively. Our results highlight the importance of angiopoietins in mesothelioma pathophysiology and pave the way for the clinical development of novel anti-angiogenic strategies.

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.

Lung adenocarcinoma (LUAD)-derived Wnts increase cancer cell proliferative/stemness potential, but whether they impact the immune microenvironment is unknown. Here we show that LUAD cells use paracrine Wnt1 signaling to induce immune resistance. In TCGA, Wnt1 correlates strongly with tolerogenic genes. In another LUAD cohort, Wnt1 inversely associates with T cell abundance. Altering Wnt1 expression profoundly affects growth of murine lung adenocarcinomas and this is dependent on conventional dendritic cells (cDCs) and T cells. Mechanistically, Wnt1 leads to transcriptional silencing of CC/CXC chemokines in cDCs, T cell exclusion and cross-tolerance. Wnt-target genes are up-regulated in human intratumoral cDCs and decrease upon silencing Wnt1, accompanied by enhanced T cell cytotoxicity. siWnt1-nanoparticles given as single therapy or part of combinatorial immunotherapies act at both arms of the cancer-immune ecosystem to halt tumor growth. Collectively, our studies show that Wnt1 induces immunologically cold tumors through cDCs and highlight its immunotherapeutic targeting., The Wnt pathway regulates anti-tumour immunity in melanoma. Here, the authors show that, in lung adenocarcinoma, secretion of the ligand Wnt1 induces immune resistance by impairing the ability of dendritic cells to cross-prime T cells, and that blocking Wnt signalling enhances rejection of tumours by acting both on the cancer and immune cells.

KRAS mutations of lung adenocarcinoma (LADC) are associated with smoking but little is known on other exposure-oncogene associations. Hypothesizing that different inciting agents may cause different driver mutations, we aimed to identify distinct molecular pathways to LADC, applying two entirely different approaches. First, we examined clinicopathologic features and genomic signatures of environmental exposures in the large LADC Campbell data set. Second, we designed a molecular mechanistic risk model of LADC (M3LADC) that links environmental exposure to incidence risk by mathematically emulating the disease process. This model was applied to incidence data of Japanese atom-bomb survivors which contains information on radiation and smoking exposure. Grouping the clinical data by driver mutations revealed two main distinct molecular pathways to LADC: one unique to transmembrane receptor-mutant patients that displayed robust signatures of radiation exposure and one shared between submembrane transducer-mutant patients and patients with no evident driver mutation that carried the signature of smoking. Consistently, best fit of the incidence data was achieved with a M3LADC with two pathways: in one LADC risk increased with radiation exposure and in the other with cigarette consumption. We conclude there are two main molecular pathways to LADC associated with different environmental exposures. Future molecular measurements in lung cancer tissue of atom-bomb survivors may allow to further test quantitatively the M3LADC-predicted link of radiation to transmembrane receptor mutations. Moreover, the developed molecular mechanistic model showed that for low doses, as relevant e.g. for medical imaging, smokers have the same radiation risk compared with never smokers.

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

Background/aim Several links between DNA replication, pluripotency and development have been recently identified. The involvement of miRNA in the regulation of cell cycle events and pluripotency factors has also gained attention. Materials and methods In the present study, we used the g:Profiler platform to analyze transcription factor binding sites, miRNA networks and protein-protein interactions to identify novel links among the aforementioned processes. Results and conclusion A complex circuitry between retinoic acid signaling, SWI/SNF components, pluripotency factors including Oct4, Sox2 and Nanog and cell cycle regulators was identified. It is suggested that the DNA replication inhibitor geminin plays a central role in this circuitry.