Non-Oncogene Addiction of KRAS-Mutant Cancers to IL-1ß via Versican and Mononuclear IKKß.

Kirsten rat sarcoma virus (KRAS)-mutant cancers are frequent, metastatic, lethal, and largely undruggable. While interleukin (IL)-1ß 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-1ß via inflammatory versican signaling to macrophage inhibitor of NF-κB kinase (IKK) ß. Human pan-cancer and experimental NF-κB reporter, transcriptome, and proteome screens reveal that KRAS-mutant tumors trigger macrophage IKKß activation and IL-1ß release via secretory versican. Tumor-specific versican silencing and macrophage-restricted IKKß deletion prevents myeloid NF-κB activation and metastasis. Versican and IKKß are mutually addicted and/or overexpressed in human cancers and possess diagnostic and prognostic power. Non-oncogene KRAS/IL-1ß addiction is abolished by IL-1ß and TLR1/2 inhibition, indicating cardinal and actionable roles for versican and IKKß in metastasis.

KRAS signaling in malignant pleural mesothelioma.

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 KRASG12D 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 KRASG12D 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.

RAS and BRAF in the foreground for non-small cell lung cancer and colorectal cancer: Similarities and main differences for prognosis and therapies.

Lung and colorectal cancer are included in the most tremendously threatening diseases in terms of incidence and death. Although they are located in completely different organs and differ in various characteristics they do share some common features, especially regarding their molecular mutational profile. Among several commonly mutated genes KRAS and BRAF are spotted to be highly associated with patient's poor disease outcome and resistance to targeted therapies mostly in liaison with other mutant activated genes. Many studies have shed light in these mechanisms for disease progression and numerous preclinical models, clinical trials and meta-analysis reports investigate the impact of specific treatments or combination of therapies. The present review is an effort to compare the mutational imprint of these genes between the two diseases and their impact in prognosis, current therapy, mechanisms of therapy resistance and future therapeutic plans and provide a spherical perspective regarding the systemic molecular profile of cancer.

Osteopontin drives KRAS-mutant lung adenocarcinoma.

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.

Osteopontin as a Link between Inflammation and Cancer: The Thorax in the Spotlight.

The glycoprotein osteopontin (OPN) possesses multiple functions in health and disease. To this end, osteopontin has beneficial roles in wound healing, bone homeostasis, and extracellular matrix (ECM) function. On the contrary, osteopontin can be deleterious for the human body during disease. Indeed, osteopontin is a cardinal mediator of tumor-associated inflammation and facilitates metastasis. The purpose of this review is to highlight the importance of osteopontin in malignant processes, focusing on lung and pleural tumors as examples.

Interleukin-1ß provided by KIT-competent mast cells is required for KRAS-mutant lung adenocarcinoma.

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-1ß 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.

Club cells form lung adenocarcinomas and maintain the alveoli of adult mice.

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.

Tobacco chemical-induced mouse lung adenocarcinoma cell lines pin the prolactin orthologue proliferin as a lung tumour promoter.

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.

Wnt1 silences chemokine genes in dendritic cells and induces adaptive immune resistance in lung adenocarcinoma.

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.

Comprehensive clinical profiling of the Gauting locoregional lung adenocarcinoma donors.

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 <45 or >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.

The expression of p-mTOR and COUP-TFII correlates with increased lymphangiogenesis and lymph node metastasis in prostate adenocarcinoma.

BACKGROUND: Mammalian target of rapamycin (mTOR) is a central regulator of major cellular processes such as growth and proliferation. Deregulated mTOR signaling is implicated in a wide spectrum of human malignancies including prostate cancer. The aim of this study is to address the role of phosphorylated mTOR (p-mTOR) in prostate adenocarcinoma-induced lymphangiogenesis and lymph node metastasis as well as to investigate its relationship with chicken ovalbumin upstream promoter transcriptional factor 2 (COUP-TFII) and the vascular endothelial growth factors A/C (VEGF A/C). METHODS: We analyzed 92 paraffin embedded specimens from patients with prostate cancer who underwent radical prostatectomy with pelvic lymph node (LN) dissection. Twenty-four of these men were pathologically assessed to have regional LN metastasis (pN1 group) and 68 with negative lymph nodes (pN0 group). Lymph vessel density was measured using anti-D2-40 and anti-LYVE-1 antibodies. The expression of p-mTOR, COUP-TFII, and VEGF A/C was also evaluated by immunohistochemistry. RESULTS: Specimens from pN1 group exhibited higher cytoplasmic p-mTOR expression compared to pN0 specimens. Mean vessel densities assessed by COUP-TFII and D2-40 were increased in pN1 tumors and positively associated with higher p-mTOR expression. Interestingly, increased expression of p-mTOR was positively associated with COUP-TFII expression in cancer cells and elevated immunoreactivity for both VEGF A and C, which in turn exhibited higher expression in pN1 group. CONCLUSIONS: Our findings suggest that increased p-mTOR and COUP-TFII expression are implicated in human prostate adenocarcinoma-induced lymphangiogenesis and LN metastasis.

IκB Kinase α Is Required for Development and Progression of KRAS-Mutant Lung Adenocarcinoma.

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 IKKß 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 IKKß, 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, IκBß, 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 IKKß 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.

Myeloid-derived interleukin-1ß drives oncogenic KRAS-NF-κΒ addiction in malignant pleural effusion.

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-1ß. Indeed, IKKα is required for the MPE-competence of KRAS-mutant tumor cells by activating non-canonical NF-κB signaling. IL-1ß fuels addiction of mutant KRAS to IKKα resulting in increased CXCL1 secretion that fosters MPE-associated inflammation. Importantly, IL-1ß-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-1ß, thereby establishing a host-to-tumor signaling circuit that culminates in inflammatory MPE development and drug resistance.

Lung carcinogenesis and fibrosis taken together: just coincidence?

PURPOSE OF REVIEW: The pathogenesis of lung cancer and pulmonary fibrotic disorders partially overlaps. This review focuses on the common features of the two disease categories, aimed at advancing our translational understanding of their pathobiology and at fostering the development of new therapies. RECENT FINDINGS: Both malignant and collagen-producing lung cells display enhanced cellular proliferation, increased resistance to apoptosis, a propensity for invading and distorting the lung parenchyma, as well as stemness potential. These characteristics are reinforced by the tissue microenvironment and inflammation seems to play an important adjuvant role in both types of disorders. SUMMARY: Unraveling the thread of the common and distinct characteristics of lung fibrosis and cancer might contribute to a more comprehensive approach of the pathobiology of both diseases and to a pathfinder for novel and personalized therapeutic strategies.

NRAS destines tumor cells to the lungs.

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.

Tumor-derived osteopontin isoforms cooperate with TRP53 and CCL2 to promote lung metastasis.

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.

A link between RelB expression and tumor progression in laryngeal cancer.

Laryngeal cancer is a frequent malignancy originating from the squamous vocal epithelium in a multi-stage fashion in response to environmental carcinogens. Although most cases can be cured by surgery and/or radiotherapy, advanced and relapsing disease is common, and biomarkers of such dismal cases are urgently needed. The cancer genome of laryngeal cancers was recently shown to feature a signature of aberrant nuclear factor (NF)-κB activation, but this finding has not been clinically exploited. We analyzed primary tumor samples of 96 well-documented and longitudinally followed patients covering the whole spectrum of laryngeal neoplasia, including 21 patients with benign laryngeal diseases, 15 patients with dysplasia, 43 patients with early-stage carcinoma, and 17 patients with locally advanced carcinoma, for immunoreactivity of RelA, RelB, P50, and P52/P100, the main NF-κB subunits that activate transcription. Results were cross-examined with indices of tumor progression and survival. Interestingly, RelB expression increased with tumor stage, grade, and local extent. Moreover, patients displaying high RelB immunoreactivity exhibited statistically significantly poorer survival compared with patients featuring low levels of RelB expression (P = 0.018 by log-rank test). Using Cox regression analyses and tumor stage, local extent, grade and RelA/RelB immunoreactivity, we develop a new score that can independently predict survival of patients with laryngeal cancer. Hence we provide a simple and affordable NF-κB-based test to predict prognosis in laryngeal cancer.

Comprehensive Evaluation of Nuclear Factor-κΒ Expression Patterns in Non-Small Cell Lung Cancer.

Nuclear factor (NF)-κB signalling is required for lung adenocarcinoma development in mice, and both of its subunits RelA and RelB were independently reported to be highly expressed in human non-small cell lung cancer (NSCLC). To comprehensively examine NF-κB expression in NSCLC, we analyzed serial sections of primary tumor samples from 77 well-documented patients (36 adenocarcinomas, 40 squamous cell carcinomas and 3 large cell carcinomas) for immunoreactivity of RelA, RelB, P50, and P52/P100. Tumor and intratumoral stroma areas were discriminated based on proliferating cell nuclear antigen immunoreactivity and inflammatory infiltration was assessed in intratumoral stroma areas. NF-κB immunoreactivity was quantified by intensity, extent, and nuclear localization and was cross-examined with tumor cell proliferation, inflammatory infiltration, and clinical-pathologic data. We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach. Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100. However, RelA and P50 were predominantly expressed in intratumoral stroma, but RelB in tumor cells. Importantly, tumor area RelA expression was correlated with the intensity of inflammatory infiltration, whereas RelB expression was identified in proliferating tumor cells. Using multiple logistic regression, we identified that tumor RelB expression was an independent predictor of lymph node metastasis, and tumor P50 was an independent predictor of TNM6 stage IIB or higher, whereas tumor RelA was an independent predictor of inflammatory infiltration. We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components. Utilizing such an approach, we identified intriguing associations between distinct NF-κB subunits and clinical and pathologic features of NSCLC.

Mast cells mediate malignant pleural effusion formation.

Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1ß, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable.

Metadherin, p50, and p65 expression in epithelial ovarian neoplasms: an immunohistochemical study.

NF-κB signaling promotes cancer progression in a large number of malignancies. Metadherin, a coactivator of the NF-κB transcription complex, was recently identified to regulate different signaling pathways that are closely related to cancer. We assessed the immunohistochemical expression of p50, p65, and metadherin in 30 ovarian carcinomas, 15 borderline ovarian tumours, and 31 benign ovarian cystadenomas. Ovarian carcinomas exhibited significantly higher expression of all 3 markers compared to benign ovarian tumours. Borderline ovarian tumours demonstrated significantly higher expression for all 3 markers compared to benign cystadenomas. Ovarian carcinomas demonstrated significantly higher expression of p50 and metadherin compared to borderline ovarian tumours, whereas no significant difference was noted in p65 expression between ovarian carcinomas and borderline ovarian tumours. There was a strong correlation with the expression levels of p50, p65, and metadherin, whereas no correlation was observed with either grade or stage. Strong p50, p65, and metadherin expression was associated with a high probability to distinguish ovarian carcinomas over borderline and benign ovarian tumours, as well as borderline ovarian tumours over benign ovarian neoplasms. A gradual increase in the expression of these molecules is noted when moving across the spectrum of ovarian carcinogenesis, from borderline ovarian tumours to epithelial carcinomas.